TAZ/WWTR1 for diagnosis and treatment of cancer

ABSTRACT

We provide an anti-TAZ agent for the treatment, prophylaxis or alleviation of cancer. We further provide a kit for detecting breast cancer in an individual or susceptibility of the individual to breast cancer comprising means for detection of TAZ expression in the individual or a sample taken from him or her as well as a method of detecting a cancer cell, the method comprising detecting modulation of expression, amount or activity of TAZ in the cell.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a 371 National Phase Entry Application ofInternational Application No. PCT/SG2008/000387 filed Oct. 6, 2008,which designates the U.S., and which claims the benefit of priorityunder 35 U.S.C. §119(e) of U.S. Provisional No. 60/977,509 filed Oct. 4,2007, and U.S. Provisional No. 61/124,119 filed Apr. 14, 2008, thecontents of which are incorporated herein by reference in theirentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 21, 2010, isnamed 049595US.txt and is 3,112 bytes in size.

FIELD

The present invention relates to the fields of medicine, cell biology,molecular biology and genetics. This invention relates to the field ofmedicine. In particular, it relates to treatment and diagnosis ofdiseases, in particular breast cancer, as well as compositions for suchuse.

BACKGROUND

TAZ, also known as WWTR1, is a 14-3-3 binding protein with a PDZ bindingmotif. TAZ/WWTR1 is known to modulate mesenchymal stem celldifferentiation

TAZ/WWTR1 was first cloned by Kanai et al. (2000). Kanai showed that TAZRNA is most highly expressed in human kidney, followed by heart,placenta and lung. Expression was detected in all tissues tested exceptthymus and peripheral blood leukocytes. Northern blot analysis of mousetissues showed transcripts expressed at highest levels in kidney, lung,liver, and heart and also in testis. Western blot analysis revealedexpression of TAZ in several epithelial and fibroblast cell lines, butnot in Jurkat T cells.

Mesenchymal stem cells are a pluripotent cell type that candifferentiate into several distinct lineages. Two key transcriptionfactors, RUNX2 (OMIM reference 600211) and peroxisomeproliferator-activated receptor-gamma (PPARG; OMIM reference 601487),drive mesenchymal stem cells to differentiate into either osteoblasts oradipocytes, respectively. Hong et al. (2005) found that TAZ/WWTR1coactivates RUNX2-dependent gene transcription while repressingPPARG-dependent gene transcription.

By modulating Taz expression in model cell lines, mouse embryonicfibroblasts, and primary mesenchymal stem cells in culture and inzebrafish in vivo, Hong et al. (2005) observed alterations in osteogenicversus adipogenic potential. Hong et al. (2005) concluded that TAZfunctions as a molecular rheostat that modulates mesenchymal stem celldifferentiation.

Murakami (2005) show that TAZ acts as a potent TBX5 coactivator thatphysically associates with TBX5 and histone acetyltransferase (HAT)proteins and mediates TBX5-dependent gene activation. Murakami (2005)suggest that TAZ plays important roles in the control of TBX5-dependentgenes during cardiac and limb development.

Hossain (2007) show that WWTR1 is critical for the integrity of renalcilia and its absence in mice leads to the development of renal cysts.Hossain (2007) concludes that Wwtr1 may represent a candidate gene forpolycystic kidney disease in humans.

In the Western world and the developed countries of Asia, breastcarcinoma is the second leading cause of cancer-related death in women(Polyak, 2001). Breast cancer tops the cancer list for women inSingapore, with 700-800 new cases being diagnosed each year (SingaporeCancer Registry Report, 2000). In the USA, 180,000 women are diagnosedannually with new cases of breast cancer (Polyak, 2001). Despite betterdiagnosis and routine screening around a quarter of the cases will diefrom their disease.

Accordingly, there is a need for improved breast cancer detection andtherapy.

SUMMARY

According to a 1^(st) aspect of the present invention, we provide ananti-TAZ agent for the treatment, prophylaxis or alleviation of cancer.

The anti-TAZ agent may be capable of down-regulating any combination ofthe expression, amount or activity of a TAZ sequence shown as GenBankaccession number NP_(—)056287, or a sequence which has at least 90%sequence identity to that sequence.

The cancer may comprise breast cancer. The cancer may comprise aninvasive or metastatic cancer such as Invasive Ductal Carcinoma (IDC).The anti-TAZ agent may downregulate TAZ by RNA interference, such as bycomprising a Small Interfering RNA (siRNA) or Short Hairpin RNA (shRNA).

The anti-TAZ agent may comprise shRNA 1 (sense oligonucleotide sequence5′-GATGAATCCGGCCTCGGCGCC-3′ (SEQ ID NO: 1)), shRNA 650 (senseoligonucleotide sequence 5′-AGAGGTACTTCCTCAATCA-3′ (SEQ ID NO: 2)) orshRNA 652 (sense oligonucleotide sequence 5′-AGGTACTTCCTCAATCACA-3′ (SEQID NO: 3)).

The anti-TAZ agent may comprise an anti-TAZ antibody, for exampleselected from the group consisting of: rabbit anti-TAZ antibody againstamino acids 160-229 of TAZ, rabbit anti-TAZ antibody (catalogue number2149S, Cell Signaling Technology, Danvers, Mass., USA), rabbitpolyclonal anti-TAZ antibody (catalogue number NB110-58359SS, NovusBiological, Littleton, Colo., USA), Mouse Monoclonal anti-TAZ [1B10](catalogue number H00006901-M12, Novus Biological, Littleton, Colo.,USA), Rabbit anti-Human TAZ Polyclonal Antibody (catalogue numberLS-B94, LifeSpan Biosciences, Inc., Seattle, Wash., USA) or p-TAZ (Ser89)-R (catalogue number sc-17610-R, Santa Cruz Biotechnology, SantaCruz, Calif., USA).

There is provided, according to a 2^(nd) aspect of the presentinvention, a nucleic acid comprising shRNA 650 (sense oligonucleotidesequence 5′-AGAGGTACTTCCTCAATCA-3′ (SEQ ID NO: 2)), shRNA 652 (senseoligonucleotide sequence 5′-AGGTACTTCCTCAATCACA-3′ (SEQ ID NO: 3)),5′-AGAGGTACTTCCTCAATCA-3′ (SEQ ID NO: 2), 5′-AGGTACTTCCTCAATCACA-3′ (SEQID NO: 3), or a complement thereof, or a nucleic acid capable ofspecifically hybridising to any such sequence.

We provide, according to a 3^(rd) aspect of the present invention, arabbit anti-TAZ antibody against amino acids 160-229 of TAZ, optionallyin combination with a polypeptide comprising a YAP sequence (GenBankAccession Number: NP_(—)006097) or a sequence having at least 90%sequence identity to that sequence, such as GST-YAP.

As a 4^(th) aspect of the present invention, there is provided anMCF10A-TAZ, MCF7-KD-1, MCF7-KD-650, MCF7-KD-652 or Hs578T-KD-652 cell orcell line, for example for use as a model for cancer, such as invasivebreast cancer.

We provide, according to a 5^(th) aspect of the present invention, a kitfor detecting breast cancer in an individual or susceptibility of theindividual to breast cancer comprising means for detection of TAZexpression in the individual or a sample taken from him or her.

The means for detection may be selected from the group consisting of: aTAZ polynucleotide or a fragment thereof; a complementary nucleotidesequence to TAZ nucleic acid or a fragment thereof; a TAZ polypeptide ora fragment thereof, or an anti-TAZ antibody for example comprising anantibody against amino acids 160-229 of TAZ, and optionally instructionsfor use. The kit may further comprise an anti-TAZ agent according to the1^(st) aspect of the invention. The kit may further comprise atherapeutic drug for treatment, prophylaxis or alleviation of breastcancer, such as comprising Tamoxifen or Herceptin.

The present invention, in a 6^(th) aspect, provides a method ofdetecting a cancer cell, the method comprising detecting modulation ofexpression, amount or activity of TAZ in the cell. The cancer maycomprise breast cancer, such as invasive or metastatic cancer such asInvasive Ductal Carcinoma (IDC). The expression of TAZ may be comparedto the expression, amount or activity of TAZ in a control cell known tobe non-cancerous.

The method may comprise detecting up-regulation of TAZ expression,amount or activity in the cell. The method may comprise detecting a TAZnucleic acid, such as by means of a probe comprising at least a portionof a nucleic acid having a sequence shown as GenBank accession numberNM_(—)015472 or a sequence having at least 90% sequence identity to sucha sequence. The method may comprise detecting a TAZ polypeptide. The TAZpolypeptide may be detected by means of an anti-TAZ antibody set outabove.

The method may comprise detecting expression of one or more proteinsselected from the group consisting of: IGFBP3, ADAMTS1, CTGF, Cyr61,FSTL1, FN1, FBN1, FBN2AXL, ITGB2, CRIM1 and Alcam.

The method may further comprise histological grading, for example usingthe Elston-Ellis modified Scarff, Bloom, Richardson grading system(Nottingham Grading System (NGS)).

A method of determining the proliferative state of a cell, ordetermining the likelihood that a cell will become invasive oraggressive, the method comprising detecting modulation of expression,amount or activity of TAZ in the cell.

In a 7^(th) aspect of the present invention, there is provided a methodof predicting a survival rate of an individual with cancer, the methodcomprising detecting modulation of expression of TAZ in a cell of theindividual

According to an 8^(th) aspect of the present invention, we provide amethod of choosing a therapy for an individual with cancer, the methodcomprising detecting modulation of expression of TAZ in a cell of theindividual choosing an appropriate therapy based on the aggressivenessof the cancer.

We provide, according to a 9^(th) aspect of the invention, a method ofdetermining the likelihood of success of a particular therapy in anindividual with a cancer, the method comprising comparing the therapywith a therapy determined by a method as set out above.

There is provided, in accordance with a 10^(th) aspect of the presentinvention, a method of manipulating a cancer cell, the method comprisingmodulating the expression, amount or activity of TAZ in the cell.

The cancer may comprise breast cancer, such as invasive or metastaticcancer such as Invasive Ductal Carcinoma (IDC). The cancer cell maybecome non-cancerous or the invasive or metastatic cancer cell becomesnon-invasive or non-metastatic as a result of the manipulation. Themethod may comprise down-regulating TAZ expression, amount or activityin the cell. The method may comprise exposing the cell to an siRNA orshRNA capable of specifically binding to TAZ. The shRNA may comprise anshRNA set out above. The method may comprise exposing the cell to ananti-TAZ antibody.

As an 11^(th) aspect of the invention, we provide a method ofmanipulating a cell, the method comprising the steps of: (a) detectingincreased TAZ expression, amount or activity in a cell; and (b) reducingthe level of TAZ in the cell.

A method of modulating the expression of TAZ, the method comprisingtargeting a TAZ target site selected from KD-1(5′-GATGAATCCGGCCTCGGCGCC-3′ (SEQ ID NO: 1)), KD-650(5′-AGAGGTACTTCCTCAATCA-3′ (SEQ ID NO: 2)) or KD-652(5′-AGGTACTTCCTCAATCACA-3′ (SEQ ID NO: 3)).

We provide, according to a 12^(th) aspect of the invention, there isprovided a method of identifying a molecule capable of binding to a TAZpolypeptide, the method comprising: (a) contacting a TAZ polypeptidewith a candidate molecule and determining whether the candidate moleculebinds to the TAZ polypeptide; or (b) a method of identifying a modulatorof TAZ, the method comprising contacting a cell with a candidatemolecule and detecting elevated or reduced expression, amount oractivity of TAZ in or of the cell.

According to a 13^(th) aspect of the present invention, we provide amethod of identifying a modulator of a TAZ polypeptide, the methodcomprising allowing TAZ to bind to a TEAD polypeptide comprising TEAD1,TEAD2, TEAD3 or TEAD4 and detecting modulation of such binding by thepresence of a candidate molecule.

There is provided, according to a 14^(th) aspect of the presentinvention, a method of identifying a molecule suitable for thetreatment, prophylaxis or alleviation of cancer, the method comprisingdetermining if a candidate molecule is an agonist or antagonist ofTAZ1/WWTR or a sequence having at least 90% sequence identity thereto.

We provide, according to a 15^(th) aspect of the present invention, useof a TAZ or a sequence having at least 90% sequence identity thereto ina method of identifying a molecule suitable for the treatment,prophylaxis or alleviation of cancer. A candidate molecule may beexposed to a TAZ polypeptide or a cell expressing a TAZ polypeptide inorder to determine if the candidate molecule is an agonist or antagonistthereof.

We provide, according to a 16^(th) aspect of the present invention, useof a TAZ polynucleotide or a sequence having at least 90% sequenceidentity thereto for the identification of a molecule suitable for thetreatment, prophylaxis or alleviation of cancer.

We provide, according to a 17^(th) aspect of the present invention, amethod of identifying an agonist or antagonist of a TAZ or a sequencehaving at least 90% sequence identity thereto, the method comprisingadministering a candidate molecule to an animal and determining whetherthe animal exhibits increased or decreased expression, amount oractivity of TAZ.

We provide, according to a 18^(th) aspect of the present invention, anexpression vector comprising a nucleic acid as set out above, such as aretroviral vector. We provide, according to a 19^(th) aspect of thepresent invention, a host cell comprising a nucleic acid as set outabove or an expression vector as set out above. We provide, according toa 20^(th) aspect of the present invention, a non-human animal comprisinga host cell as set out above.

We provide, according to a 21^(st) aspect of the present invention, amethod of treatment, prophylaxis or alleviation of a cancer in anindividual, the method comprising modulating the expression, amount oractivity of a TAZ in a cell of an individual. The expression, amount oractivity of TAZ may be decreased in a breast cell of the individual.

We provide, according to a 22^(nd) aspect of the present invention, amethod of diagnosis of a cancer or susceptibility to cancer in anindividual, the method comprising detecting modulation of expression,amount or activity of TAZ in a cell of the individual.

We provide, according to a 23^(rd) aspect of the present invention, amethod of prognosis of an individual with cancer, the method comprisingdetecting modulation of expression, amount or activity of TAZ in a cellof the individual.

We provide, according to a 24^(th) aspect of the present invention, amethod of determining whether a tumour in an individual is, or is likelyto be, an invasive or metastatic tumour, the method comprising detectingmodulation of expression, amount or activity of TAZ in a tumour cell ofthe individual.

We provide, according to a 25^(th) aspect of the present invention, amethod of treatment, prophylaxis or alleviation of cancer in anindividual, the method comprising detecting modulation of expression,amount or activity of TAZ in a cell of the individual and administeringan appropriate therapy to the individual based on the aggressiveness ofthe tumour. The cancer may comprise breast cancer, such as invasive ormetastatic cancer such as Invasive Ductal Carcinoma (IDC). Thediagnosis, prognosis or choice of therapy may be further determined byassessing the size of the tumour, or the lymph node stage, or both,optionally together or in combination with other risk factors. Thediagnosis, prognosis or choice of therapy may be further determined byassessing the oestrogen receptor (ER) status of the tumour.

We provide, according to a 26^(th) aspect of the present invention, TAZfor use in a method of treatment, prophylaxis or alleviation of acancer, for example breast cancer, in an individual.

We provide, according to a 27^(th) aspect of the present invention, amolecule, agonist or antagonist of a TAZ polypeptide identified by amethod or use as set out above.

We provide, according to a 28^(th) aspect of the present invention,molecule capable of modulating, such as down-regulating, the expressionof a TAZ for use in the treatment, prophylaxis or alleviation of cancer.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A and FIG. 1B. TAZ is preferentially overexpressed in highlyinvasive breast cancer cells.

FIG. 1A. Lysates derived from 11 breast cancer cell lines are analyzedby western blot using anti-TAZ antibodies which also reacted well withYAP (upper panels) or anti-YAP antibodies (lower panels). The levels ofactin as detected by anti-actin antibodies are used as loading controls.

FIG. 1B. The expression levels of TAZ and YAP are quantified in threeindependent experiments and the averages are presented as arbitraryunits relative to actin.

FIG. 2A, FIG. 2B and FIG. 2C. TAZ promotes migration and invasion ofMCF10A cells.

FIG. 2A. the levels of TAZ in MCF10A cells transduced with retrovirusexpressing EGFP (lane 1), TAZ (lane 2) or Flag-TAZ (lane 3) are assessedby western blot. The levels of TAZ in Hs578T (lane 4) and BT-549 (lane5) cells are assessed as comparisons.

FIG. 2B. wound-healing migration assay for MCF10A cells expressing EGFP(left panels) and MCF10A cells expressing TAZ (right panels). Thehealing of wounds by migrated cells at time 0, 14 hr and 24 hr isimaged. MCF10A cells expressing TAZ have more motile and spindle-shapedfibroblast-like appearance and migrate faster than MCF10A cellsexpressing EGFP.

FIG. 2C. The migration (left panel) and invasion (right panel) of MCF10Acells expressing EGFP and TAZ are assessed by transwell assays. Columns,mean of three independent experiments; bars, SEM.

FIG. 3A. FIG. 3B and FIG. 3C. TAZ knockdown in MCF7 cells enhancesepithelial morphology and in Hs578T cells suppresses cell migration andinvasion.

FIG. 3A. The expression levels of TAZ in MCF7 (left panel) and Hs578T(right panel) cells transduced with the vector (lane 1) and variousshRNAs (lane 2-6) targeting different sites of TAZ mRNA are assessed bywestern blot. shRNA-652 is most potent in suppressing TAZ expression inboth MCF7 and Hs578T cells.

FIG. 3B. TAZ knockdown in MCF7 cells results in clusters of moredensely-packed and compact sheets of cells. When plated at low to mediumdensities, both MCF7-KD-715 cells and MCF7-KD-652 cells grew as clustersof cells. However, the cell density of the clusters is obviouslyenhanced in the MCF7-KD-652 cells (panel b) as compared to MCF7-KD-715cells (panel a). Scanning electron microscopy revealed that the spacebetween cells is reduced in MCF7-KD-652 cells (panel d) as compared toMCF7-KD-715 cells (panel c). This resulted in the appearance of moretightly aligned/packed and compact epithelia when TAZ expression isknocked-down.

FIG. 3C. TAZ knockdown in Hs578T cells suppresses cell migration.Wound-healing migration assay for Hs578T-KD-715 (left panels) andHs578T-KD-652 (right panels) cells is performed. The healing of woundsby migrated cells at time 0, 6 hr and 12 hr is imaged. Hs578T-KD-652cells with TAZ knockdown have much reduced motility as compared toHs578T-KD-715 cells which have similar migration as parental andvector-transduced Hs578T cells. D, the migration (left panel) andinvasion (right panel) of Hs578T-KD-715 and Hs578T-KD-652 cells areassessed by transwell assays. Columns, mean of three independentexperiments; bars, SEM.

FIG. 4A. FIG. 4B and FIG. 4C. TAZ knockdown in MCF7 cells suppressesanchorage-independent growth in soft-agar and tumorigenesis in nudemice.

FIG. 4A. Soft-agar growth of MCF7-KD-715 (upper panel) and MCF7-KD-652(lower panel) cells is assessed and photographed.

FIG. 4. The appearance of live colonies in soft-agar of MCF7-KD-715(left panel) and MCF7-KD-652 (right panel) cells is photographed athigher amplification.

FIG. 4C. Tumor formation of MCF7-KD-715 (right side) and MCF7-KD-652(left side) cells in the thigh (left panel) or the fat pad (right panel)of nude mice are assessed and photographed.

FIG. 5A and FIG. 5B. TAZ is overexpressed in invasive (infiltrating)ductal carcinomas (IDCs).

FIG. 5A. Characterization of rabbit anti-TAZ antibodies. Lysate derivedfrom the indicated cells are analyzed by western blot usingaffinity-purified rabbit antibodies raised against fragment (amino acids160-229) of TAZ in the absence (left panel) or the presence of 100× ofrecombinant YAP fragment (amino acids 206-262) corresponding to the TAZantigen region. Although the antibodies cross-reacted with YAP (leftpanel), they recognized specifically TAZ in the presence of excessamount of recombinant YAP fragment.

FIG. 5B. TAZ is overexpressed in invasive ductal carcinomas (IDC).Normal breast tissues (left panels) or breast cancer tissues (IDC)(right panels) are either stained with TAZ antibody (preincubated with100 folds excess of recombinant YAP fragment) or cytokeratin antibody asa control. TAZ is overexpressed in IDC but not in normal breast tissue.

FIG. 6A and FIG. 6B. Quantification of tumors excised from mice.

FIG. 6A. Tumors excised from the fat pad of one pair of mice injectedwith MCF-KD-715 and MCF7-KD-652 cells, respectively, are pictured.

FIG. 6B. Quantitation of tumor weights. The tumors excised from the fatpad of mice injected with MCF-KD-715 and MCF7-KD-652 cells are weighedand presented. Bars, SEM.

FIG. 7A, FIG. 7B and FIG. 7C. Expression of mouse TAZ (mTAZ) in TAZknocked-down cells, MCF7-KD-652, significantly restores the growth ofMCF7-KD-652 cells in soft agar. MCF7-KD-715 and MCF7-KD-652 cells areinfected with pBABE hygromycin retroviral vector (control) or theretroviral vector expressing mTAZ (Flag-mTAZ). After double selection inthe presence of puromycin (1 μg/ml) and hygromycin (500 μg/ml), cellsare analyzed by immunoblot analysis (FIG. 7A) and growth in soft agarfor two weeks (FIG. 7B). TAZ, YAP and Flag-mTAZ are indicated.Quantitation of the colony numbers of cells in the soft-agar assay fromthree independent experiments is shown in FIG. 7C. Bars, SEM.

FIG. 8A and FIG. 8B. Retention of TAZ and YAP by immobilized C-terminalregion of TEAD 1-4. FIG. 8A and FIG. 8B show that TAZ interacts withtranscriptional factors (TEAD1, 2, 3 and 4).

FIG. 9 is a figure showing the identification of residues in TAZ thatare important for interaction with TEAD.

FIG. 10 is a figure showing results of experiments testing the abilityof TAZ mutants to accumulate in the nucleus.

FIG. 11 is a figure showing results of experiments testing the abilityof TAZ mutants in driving anchorage-independent growth.

FIG. 12 is a figure showing results of RT-PCR experiments showing theexpression of secreted proteins and surface membrane proteins.

DETAILED DESCRIPTION

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA and immunology, which are within thecapabilities of a person of ordinary skill in the art. Such techniquesare explained in the literature. See, for example, J. Sambrook, E. F.Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual,Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel,F. M. et al. (1995 and periodic supplements; Current Protocols inMolecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York,N.Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation andSequencing: Essential Techniques, John Wiley & Sons; J. M. Polak andJames O'D. McGee, 1990, In Situ Hybridization: Principles and Practice;Oxford University Press; M. J. Gait (Editor), 1984, OligonucleotideSynthesis: A Practical Approach, Irl Press; D. M. J. Lilley and J. E.Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesisand Physical Analysis of DNA Methods in Enzymology, Academic Press;Using Antibodies: A Laboratory Manual: Portable Protocol NO. I by EdwardHarlow, David Lane, Ed Harlow (1999, Cold Spring Harbor LaboratoryPress, ISBN 0-87969-544-7); Antibodies: A Laboratory Manual by Ed Harlow(Editor), David Lane (Editor) (1988, Cold Spring Harbor LaboratoryPress, ISBN 0-87969-314-2), 1855. Handbook of Drug Screening, edited byRamakrishna Seethala, Prabhavathi B. Fernandes (2001, New York, N.Y.,Marcel Dekker, ISBN 0-8247-0562-9); and Lab Ref: A Handbook of Recipes,Reagents, and Other Reference Tools for Use at the Bench, Edited JaneRoskams and Linda Rodgers, 2002, Cold Spring Harbor Laboratory, ISBN0-87969-630-3. Each of these general texts is herein incorporated byreference.

Use of TAZ in the Treatment and Diagnosis of Breast Cancer

The present invention is based on the demonstration, for the first time,that TAZ plays a role in cancer.

Specifically, we show that TAZ plays a critical role in migration,invasion and tumorigenesis of breast cancer cells. We show in theExamples that TAZ is prominently expressed in human breast cancer celllines where its expression levels generally correlate with theinvasiveness and/or aggressiveness of the cancer cells. High levels ofTAZ are detected in highly invasive breast cancer cell lines such asHs578T, BT549, MDA-MB-453 and MDA-MB231. Lower levels of TAZ aredetected in less invasive breast cancer cell lines such as MCF10A, BT20,MCF7, MDA-MB-453, ZR75.1 and BT474.

Accordingly, TAZ may be used as a marker for detection of breast cancer,including basal-like, triple negative and BRCA1-mutated cancer types.The level of TAZ expression may be used as an indicator of cancer, inparticular breast cancer such as metastatic, aggressive or invasivebreast cancer. The level of TAZ expression may also be used as anindicator of likelihood of such a cancer. We therefore provide formethods of diagnosis or detection of a cancer, particularly breastcancer. We further provide methods of diagnosis and detection of theaggressiveness or invasiveness or the metastatic state, or anycombination of these, of such a cancer. The methods may compriseanalysis of protein levels (e.g., immunohistochemistry) or RNA levels(e.g., by in situ hybridisation). Such diagnostic and detection methodsare described in further detail below.

We show that over-expression of TAZ in immortalized but not transformedMCF10A cells promotes cell proliferation, cell migration and invasion.shRNA mediated knock-down of TAZ in Hs578-t and MCF7 cells suppressescell migration and invasion. Anchorage-dependent growth in soft-agar andtumorigenesis in vivo of MCF7 cells is suppressed by TAZ knockdown.Culture media harvested from MCF10A cells over-expressing TAZ promotescell migration. Over-expression of TAZ therefore causes a cancerousphenotype.

Accordingly, we provide for methods of treatment or prophylaxis of anindividual suffering from cancer. Restoration of TAZ levels to those innormal tissue may also be used as a means of restoring normal functionof breast cells. We therefore provide for the use of TAZ nucleic acidsand polypeptides for the treatment of cancers, including breast cancer.Our methods may be used for treatment or prophylaxis of breast cancer orinvasive cancer such as invasive breast cancer.

We show in the Examples and FIG. 1 that TAZ is over-expressed ininvasive breast cancer lines belonging to the basal-like, triplenegative or BRCA1-mutated types. FIG. 1B shows that three (Hs578T,BT-549, and MDA-MB-435S) of the four highly invasive cancer cell linesexhibit high levels of TAZ expression with MDA-MB-231 cells expressingmoderate levels.

These four cell lines are shown in Neve et al (2006) to correspond tobasal-like or basal B cancer type and represents an invasive breastcancer cell type. This cell type is also referred to as “invasive celltype” or “basal cell type” (BRCA1-mutated is also known asBRCA1-negative). Accordingly, TAZ may be used as a marker for detectionof invasive cell type or basal cell type breast cancers.

Unlike ER+ or Her2+ breast cancers, there is no targeted therapy forinvasive breast cancers belonging to any of these three breast cancertypes. We therefore disclose for the first time a method of treating, ina targeted fashion, an invasive cell type, a basal-like breast cancer, atriple negative breast cancer and a BRCA1-mutated breast cancer type.

We further provide for the use of TAZ in screening for drugs againstcancer, particularly breast cancer, more particularly invasive breastcancer. Such screens may involve detecting the modulation of bindingbetween TAZ and TEAD1/2/3/4 by the presence of a candidate molecule.

We provide for a method of identifying a molecule for the treatment orprophylaxis of cancer, including breast cancer such as invasive breastcancer, the method comprising identifying a modulator of an activity orexpression of TAZ.

We show in the Examples that TAZ interacts with TEAD1, TEAD2, TEAD3 andTEAD4, and that this interaction is essential for nuclear accumulationof TAZ and oncogenesis.

Accordingly, we provide for a method of identifying a molecule for thetreatment or prophylaxis of cancer, including breast cancer such asinvasive breast cancer, the method comprising detecting an effect of acandidate molecule on the binding between TAZ and TEAD1, TEAD2, TEAD3and/or TEAD4. A screen for small molecule inhibitors of TAZ-TEAD bindingmay be conducted on a library for example.

Alternatively, or in addition, rational design may be employed toproduce candidate inhibitors of a TAZ-TEAD interaction. Thus, forexample, a peptide from a TAZ binding region of a TEAD (including TEAD1,TEAD2, TEAD3 or TEAD4) may be designed. Similarly, a peptide from a TEADbinding region of a TAZ (including a TEAD1, TEAD2, TEAD3 or TEAD4binding region) may be designed. Such a peptide could include mutatedpositions 52 and 53 of TAZ, both of which are shown in the Examples asbeing important in the binding between TAZ and TEAD.

Putative inhibitors (or candidate inhibitors identified in a screen) maybe tested using a number of assays, including a nuclear accumulationassay or a soft agar assay, both of which are described in the Examples.

We further provide for the treatment or prophylaxis of cancer byinterfering with or disrupting a TAZ-TEAD interaction. This may beachieved by various means, for example, by introducing a modulator ofTAZ, such as a molecule identified from a screen or design describedabove, to a patient in need thereof.

We show in the Examples that TAZ induces or up-regulates the expressionof 8 secreted proteins (IGFBP3, ADAMTS1, CTGF, Cyr61, FSTL1, FN1, FBN1and FBN2) as well as 4 surface membrane proteins (AXL, ITGB2, CRIM1, andAlcam). Each of these proteins may therefore be used as markers of theoncogenic state. Accordingly, we provide for the detection of a cancercell or an oncogenic cell or a metastatic cell, the method comprisingdetecting the expression of a polypeptide selected from the groupconsisting of: IGFBP3, ADAMTS1, CTGF, Cyr61, FSTL1, FN1, FBN1, FBN2AXL,ITGB2, CRIM1 and Alcam. We also provide for the treatment of cancer, themethod comprising modulating the activity or a polypeptide selected fromthe group consisting of: IGFBP3, ADAMTS1, CTGF, Cyr61, FSTL1, FN1, FBN1,FBN2AXL, ITGB2, CRIM1 and Alcam.

Cells over- and under-expressing TAZ, as well as tissues, organs andorganisms comprising these may be used as models for cancer or inscreens for anti-cancer agents.

TAZ

TAZ is also referred to as Transcriptional Coactivator with PDZ-BindingMotif or WWTR1. It maps to gene map locus 3q24.

TAZ was first cloned by Kanai et al. (2000) from a HeLa cDNA expressionlibrary by screening for 14-3-3-binding proteins (OMIM reference605066), followed by 5-prime RACE. The deduced protein contains 400amino acids and has an apparent molecular mass of 45 kD in HeLa cells.It has a putative 14-3-3 protein-binding motif in its N terminus, acentral WW domain, and a putative 2-stranded coiled-coil and aPDZ-binding motif in its C terminus.

TAZ shares 91% sequence identity with the mouse Taz protein and 45%identity with YAP (OMIM reference 606608). Northern blot analysisrevealed highest expression of a 6-kb transcript in kidney, followed byheart, placenta, and lung. Expression was detected in all tissues testedexcept thymus and peripheral blood leukocytes. Northern blot analysis ofmouse tissues showed a 5.5-kb transcript expressed at highest levels inkidney, lung, liver, and heart; a 2.2-kb transcript was detected intestis. Western blot analysis revealed expression of TAZ in severalepithelial and fibroblast cell lines, but not in Jurkat T cells.

Kanai et al. (2000) characterized murine Taz. They found that theinteraction of Taz with rat 14-3-3 required Taz phosphorylation on aspecific serine residue. Phosphorylation reduced Taz transcriptionalcoactivation by inducing nuclear export through interaction with 14-3-3.The C-terminal PDZ-binding domain localized Taz to discrete nuclear fociand was required for Taz-stimulated gene transcription. The PDZ-bindingdomain also mediated Taz interaction with Nherf2 (OMIM reference606553).

Mesenchymal stem cells are a pluripotent cell type that candifferentiate into several distinct lineages. Two key transcriptionfactors, RUNX2 (OMIM reference 600211) and peroxisomeproliferator-activated receptor-gamma (PPARG; OMIM reference 601487),drive mesenchymal stem cells to differentiate into either osteoblasts oradipocytes, respectively. Hong et al. (2005) found that TAZ, a14-3-3-binding protein, coactivates RUNX2-dependent gene transcriptionwhile repressing PPARG-dependent gene transcription. By modulating Tazexpression in model cell lines, mouse embryonic fibroblasts, and primarymesenchymal stem cells in culture and in zebrafish in vivo, Hong et al.(2005) observed alterations in osteogenic versus adipogenic potential.Hong et al. (2005) concluded that TAZ functions as a molecular rheostatthat modulates mesenchymal stem cell differentiation.

Murakami et al. (2005) found that TAZ was a potent TBX5 (OMIM reference601620) transactivator. TAZ associated with TBX5 and stimulatedTBX5-dependent promoters by interacting with the histoneacetyltransferases p300 (EP300; OMIM reference 602700) and PCAF (OMIMreference 602303). TBX5 with Holt-Oram syndrome (HOS; OMIM reference142900)-associated truncation mutations could not be stimulated by TAZ,but TBX5 with HOS-associated point mutations was unimpaired in itsability to respond to TAZ.

By radiation hybrid analysis, Kanai et al. (2000) mapped the TAZ gene tochromosome 3q24.

Where the term “TAZ” is used, this should be taken to refer to any TAZsequence, including a TAZ protein or a TAZ nucleic acid and anyfragment, variant homologue, derivative, variant thereof.

The properties and activities of TAZ are described in this document, forexample, in the references.

TAZ Polypeptides

The methods and compositions described here make use of TAZpolypeptides, which are described in detail below.

As used here, the term “TAZ polypeptide” is intended to refer to asequence having GenBank Accession number NP_(—)056287, NP_(—)598545,NP_(—)001032785, XP_(—)871504, NP_(—)001020040. A “TAZ polypeptide” maycomprise or consist of a human TAZ polypeptide, such as the sequencehaving accession number NP_(—)056287.

Homologues variants and derivatives thereof of any, some or all of thesepolypeptides are also included. For example, TAZ may include GenBankAccession Number AJ299430.

TAZ polypeptides may be used for a variety of means, for example,administration to an individual suffering from, or suspected to besuffering from, breast cancer, for the treatment thereof. They may alsobe used for production or screening of anti-TAZ agents such as specificTAZ binding agents, in particular, anti-TAZ antibodies. These aredescribed in further detail below. The expression of TAZ polypeptidesmay be detected for diagnosis or detection of cancer, in particularbreast cancer.

A “polypeptide” refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds, i.e., peptide isosteres. “Polypeptide” refers to both shortchains, commonly referred to as peptides, oligopeptides or oligomers,and to longer chains, generally referred to as proteins. Polypeptidesmay contain amino acids other than the 20 gene-encoded amino acids.

“Polypeptides” include amino acid sequences modified either by naturalprocesses, such as post-translational processing, or by chemicalmodification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.

Polypeptides may be branched as a result of ubiquitination, and they maybe cyclic, with or without branching. Cyclic, branched and branchedcyclic polypeptides may result from posttranslation natural processes ormay be made by synthetic methods. Modifications include acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-inking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-inks, formation of cystine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination. See, for instance, Proteins—Structure and MolecularProperties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, NewYork, 1993 and Wold, F., Posttranslational Protein Modifications:Perspectives and Prospects, pgs. 1-12 in Posttranslational CovalentModification of Proteins, B. C. Johnson, Ed., Academic Press, New York,1983; Seifter et al., “Analysis for protein modifications and nonproteincofactors”, Meth Enzymol (1990) 182:626-646 and Rattan et al., “ProteinSynthesis: Posttranslational Modifications and Aging”, Ann NY Acad Sci(1992) 663:48-62.

The term “polypeptide” includes the various synthetic peptide variationsknown in the art, such as a retroinverso D peptides. The peptide may bean antigenic determinant and/or a T-cell epitope. The peptide may beimmunogenic in vivo. The peptide may be capable of inducing neutralisingantibodies in vivo.

As applied to TAZ, the resultant amino acid sequence may have one ormore activities, such as biological activities in common with a TAZpolypeptide, for example a human TAZ polypeptide. For example, a TAZhomologue may have a increased expression level in breast cancer cellscompared to normal breast cells. In particular, the term “homologue”covers identity with respect to structure and/or function providing theresultant amino acid sequence has TAZ activity. With respect to sequenceidentity (i.e. similarity), there may be at least 70%, such as at least75%, such as at least 85%, such as at least 90% sequence identity. Theremay be at least 95%, such as at least 98%, sequence identity. Theseterms also encompass polypeptides derived from amino acids which areallelic variations of the TAZ nucleic acid sequence.

Where reference is made to the “activity” or “biological activity” of apolypeptide such as TAZ, these terms are intended to refer to themetabolic or physiological function of TAZ, including similar activitiesor improved activities or these activities with decreased undesirableside effects. Also included are antigenic and immunogenic activities ofthe TAZ. Examples of such activities, and methods of assaying andquantifying these activities, are known in the art, and are described indetail elsewhere in this document.

For example, such activities may include any one or more of thefollowing: binding to SLC9A3R2, binding to YWHA, binding to 14-3-3,co-activation of RUNX2-dependent gene transcription, transactivation ofTBX5 by TAZ, etc, as described in more detail below.

Other TAZ Polypeptides

TAZ variants, homologues, derivatives and fragments are also of use inthe methods and compositions described here.

The terms “variant”, “homologue”, “derivative” or “fragment” in relationto TAZ include any substitution of, variation of, modification of,replacement of, deletion of or addition of one (or more) amino acid fromor to a sequence. Unless the context admits otherwise, references to“TAZ” includes references to such variants, homologues, derivatives andfragments of TAZ.

As used herein a “deletion” is defined as a change in either nucleotideor amino acid sequence in which one or more nucleotides or amino acidresidues, respectively, are absent. As used herein an “insertion” or“addition” is that change in a nucleotide or amino acid sequence whichhas resulted in the addition of one or more nucleotides or amino acidresidues, respectively, as compared to the naturally occurringsubstance. As used herein “substitution” results from the replacement ofone or more nucleotides or amino acids by different nucleotides or aminoacids, respectively.

TAZ polypeptides as described here may also have deletions, insertionsor substitutions of amino acid residues which produce a silent changeand result in a functionally equivalent amino acid sequence. Deliberateamino acid substitutions may be made on the basis of similarity inpolarity, charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues. For example, negatively chargedamino acids include aspartic acid and glutamic acid; positively chargedamino acids include lysine and arginine; and amino acids with unchargedpolar head groups having similar hydrophilicity values include leucine,isoleucine, valine, glycine, alanine, asparagine, glutamine, serine,threonine, phenylalanine, and tyrosine.

Conservative substitutions may be made, for example according to thetable below. Amino acids in the same block in the second column and inthe same line in the third column may be substituted for each other:

ALIPHATIC Non-polar G A P I L V Polar - uncharged C S T M N Q Polar -charged D E K R AROMATIC H F W Y

TAZ polypeptides may further comprise heterologous amino acid sequences,typically at the N-terminus or C-terminus, such as the N-terminus.Heterologous sequences may include sequences that affect intra orextracellular protein targeting (such as leader sequences). Heterologoussequences may also include sequences that increase the immunogenicity ofthe TAZ polypeptide and/or which facilitate identification, extractionand/or purification of the polypeptides. Another heterologous sequencethat may be used is a polyamino acid sequence such as polyhistidinewhich may be N-terminal. A polyhistidine sequence of at least 10 aminoacids, such as at least 17 amino acids but fewer than 50 amino acids maybe employed.

The TAZ polypeptides may be in the form of the “mature” protein or maybe a part of a larger protein such as a fusion protein. It is oftenadvantageous to include an additional amino acid sequence which containssecretory or leader sequences, pro-sequences, sequences which aid inpurification such as multiple histidine residues, or an additionalsequence for stability during recombinant production.

TAZ polypeptides as described here are advantageously made byrecombinant means, using known techniques. However they may also be madeby synthetic means using techniques well known to skilled persons suchas solid phase synthesis. Such polypeptides may also be produced asfusion proteins, for example to aid in extraction and purification.Examples of fusion protein partners include glutathione-S-transferase(GST), 6×His (SEQ ID NO: 4), GAL4 (DNA binding and/or transcriptionalactivation domains) and □-galactosidase. It may also be convenient toinclude a proteolytic cleavage site between the fusion protein partnerand the protein sequence of interest to allow removal of fusion proteinsequences, such as a thrombin cleavage site. The fusion protein may beone which does not hinder the function of the protein of interestsequence.

The TAZ polypeptides may be in a substantially isolated form. This termis intended to refer to alteration by the hand of man from the naturalstate. If an “isolated” composition or substance occurs in nature, ithas been changed or removed from its original environment, or both. Forexample, a polynucleotide, nucleic acid or a polypeptide naturallypresent in a living animal is not “isolated,” but the samepolynucleotide, nucleic acid or polypeptide separated from thecoexisting materials of its natural state is “isolated”, as the term isemployed herein.

It will however be understood that the TAZ protein may be mixed withcarriers or diluents which will not interfere with the intended purposeof the protein and still be regarded as substantially isolated. A TAZpolypeptide may also be in a substantially purified form, in which caseit will generally comprise the protein in a preparation in which morethan 90%, for example, 95%, 98% or 99% of the protein in the preparationis a TAZ polypeptide.

By aligning TAZ sequences from different species, it is possible todetermine which regions of the amino acid sequence are conserved betweendifferent species (“homologous regions”), and which regions vary betweenthe different species (“heterologous regions”).

The TAZ polypeptides may therefore comprise a sequence which correspondsto at least part of a homologous region. A homologous region shows ahigh degree of homology between at least two species. For example, thehomologous region may show at least 70%, at least 80%, at least 90% orat least 95% identity at the amino acid level using the tests describedabove. Peptides which comprise a sequence which corresponds to ahomologous region may be used in therapeutic strategies as explained infurther detail below. Alternatively, the TAZ peptide may comprise asequence which corresponds to at least part of a heterologous region. Aheterologous region shows a low degree of homology between at least twospecies.

TAZ Homologues

The TAZ polypeptides disclosed for use include homologous sequencesobtained from any source, for example related viral/bacterial proteins,cellular homologues and synthetic peptides, as well as variants orderivatives thereof. Thus polypeptides also include those encodinghomologues of TAZ from other species including animals such as mammals(e.g. mice, rats or rabbits), especially primates, more especiallyhumans. More specifically, homologues include human homologues.

In the context of this document, a homologous sequence is taken toinclude an amino acid sequence which is at least 15, 20, 25, 30, 40, 50,60, 70, 80 or 90% identical, such as at least 95 or 98% identical at theamino acid level, for example over at least 50 or 100, 200, 300, 400 or500 amino acids with the sequence of a relevant TAZ sequence.

In particular, homology should typically be considered with respect tothose regions of the sequence known to be essential for protein functionrather than non-essential neighbouring sequences. This is especiallyimportant when considering homologous sequences from distantly relatedorganisms.

Although homology can also be considered in terms of similarity (i.e.amino acid residues having similar chemical properties/functions), inthe context of the present document homology may be expressed in termsof sequence identity.

Homology comparisons can be conducted by eye, or more usually, with theaid of readily available sequence comparison programs. These publiclyand commercially available computer programs can calculate % identitybetween two or more sequences.

% identity may be calculated over contiguous sequences, i.e. onesequence is aligned with the other sequence and each amino acid in onesequence directly compared with the corresponding amino acid in theother sequence, one residue at a time. This is called an “ungapped”alignment. Typically, such ungapped alignments are performed only over arelatively short number of residues (for example less than 50 contiguousamino acids).

Although this is a very simple and consistent method, it fails to takeinto consideration that, for example, in an otherwise identical pair ofsequences, one insertion or deletion will cause the following amino acidresidues to be put out of alignment, thus potentially resulting in alarge reduction in % homology when a global alignment is performed.Consequently, most sequence comparison methods are designed to produceoptimal alignments that take into consideration possible insertions anddeletions without penalising unduly the overall homology score. This isachieved by inserting “gaps” in the sequence alignment to try tomaximise local identity or similarity.

However, these more complex methods assign “gap penalties” to each gapthat occurs in the alignment so that, for the same number of identicalamino acids, a sequence alignment with as few gaps aspossible—reflecting higher relatedness between the two comparedsequences—will achieve a higher score than one with many gaps. “Affinegap costs” are typically used that charge a relatively high cost for theexistence of a gap and a smaller penalty for each subsequent residue inthe gap. This is the most commonly used gap scoring system. High gappenalties will of course produce optimised alignments with fewer gaps.Most alignment programs allow the gap penalties to be modified. However,the default values may be used when using such software for sequencecomparisons. For example when using the GCG Wisconsin Bestfit package(see below) the default gap penalty for amino acid sequences is −12 fora gap and −4 for each extension.

Calculation of maximum % homology therefore firstly requires theproduction of an optimal alignment, taking into consideration gappenalties. A suitable computer program for carrying out such analignment is the GCG Wisconsin Bestfit package (University of Wisconsin,U.S.A; Devereux et al., 1984, Nucleic Acids Research 12:387). Examplesof other software than can perform sequence comparisons include, but arenot limited to, the BLAST package (see Ausubel et al., 1999 ibid—Chapter18), FASTA (Altschul et al., 1990, J. Mol. Biol., 403-410) and theGENEWORKS suite of comparison tools. Both BLAST and FASTA are availablefor offline and online searching (see Ausubel et al., 1999 ibid, pages7-58 to 7-60). The GCG Bestfit program may be used.

Although the final % homology can be measured in terms of identity, thealignment process itself is typically not based on an all-or-nothingpair comparison. Instead, a scaled similarity score matrix is generallyused that assigns scores to each pairwise comparison based on chemicalsimilarity or evolutionary distance. An example of such a matrixcommonly used is the BLOSUM62 matrix—the default matrix for the BLASTsuite of programs. GCG Wisconsin programs generally use either thepublic default values or a custom symbol comparison table if supplied(see user manual for further details). The public default values for theGCG package may be used, or in the case of other software, the defaultmatrix, such as BLOSUM62.

Once the software has produced an optimal alignment, it is possible tocalculate % homology, such as % sequence identity. The softwaretypically does this as part of the sequence comparison and generates anumerical result.

The terms “variant” or “derivative” in relation to amino acid sequencesincludes any substitution of, variation of, modification of, replacementof, deletion of or addition of one (or more) amino acids from or to thesequence providing the resultant amino acid sequence retainssubstantially the same activity as the unmodified sequence, such ashaving at least the same activity as the TAZ polypeptides.

Polypeptides having the TAZ amino acid sequence disclosed here, orfragments or homologues thereof may be modified for use in the methodsand compositions described here. Typically, modifications are made thatmaintain the biological activity of the sequence. Amino acidsubstitutions may be made, for example from 1, 2 or 3 to 10, 20 or 30substitutions provided that the modified sequence retains the biologicalactivity of the unmodified sequence. Alternatively, modifications may bemade to deliberately inactivate one or more functional domains of thepolypeptides described here. Amino acid substitutions may include theuse of non-naturally occurring analogues, for example to increase bloodplasma half-life of a therapeutically administered polypeptide.

TAZ Fragments

Polypeptides for use in the methods and compositions described here alsoinclude fragments of the full length sequence of any of the TAZpolypeptides identified above. Fragments may comprise at least oneepitope. Methods of identifying epitopes are well known in the art.Fragments will typically comprise at least 6 amino acids, such as atleast 10, 20, 30, 50 or 100 amino acids.

Included are fragments comprising or consisting of, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170,175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240,245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310,315 or more residues from a relevant TAZ amino acid sequence.

We further describe peptides comprising a portion of a TAZ polypeptideas described here. Thus, fragments of TAZ and its homologues, variantsor derivatives are included. The peptides may be between 2 and 200 aminoacids, such as between 4 and 40 amino acids in length. The peptide maybe derived from a TAZ polypeptide as disclosed here, for example bydigestion with a suitable enzyme, such as trypsin. Alternatively thepeptide, fragment, etc may be made by recombinant means, or synthesisedsynthetically.

A fragment of TAZ may comprise a TAZ fragment of a sequence havingaccession number NP_(—)056287. A fragment of TAZ may comprise a sequenceof TAZ which shares low similarity or identity to YAP (Sudol et al,1995; GenBank Accession Number: NP_(—)006097). For example a TAZfragment may comprise amino acid residues 160-229 of TAZ, i.e:

(SEQ ID NO: 5) n qplnhmnlhp aysstpvpqr smavsqpnlv mnhqhqqqmapstlsqqnhp tqnppaglms mpnalttqq

Accordingly, such a TAZ fragment may be used to generate probes topreferentially detect TAZ expression, for example, through antibodiesgenerated against such fragments. These antibodies would be expected tobind specifically to TAZ, and are useful in the methods of diagnosis andtreatment disclosed here.

Other fragments of TAZ may comprise fragments comprising residue 52 orresidue 53 of TAZ, i.e., F52 or F53, or comprising mutations at one orboth of these positions. In this regard, the Examples disclose thatpositions 52 and 53 of TAZ, both of which are occupied by phenylalanineresidues, are important in the binding between TAZ and TEAD. They arealso shown to be important for nuclear accumulation of TAZ and itsoncogenic potential. Accordingly, we disclose fragments of TAZ whichcomprise a mutation at F52 or a mutation at F53, or both.

Such fragments of TAZ may suitably be provided in the form of peptides,which may be used as anti-TAZ peptides. Accordingly, we disclosepeptides comprising sequences of TAZ which flank either or both of thesepositions. The peptides may be of any suitable length, such as between 5to 40 (or more) residues of TAZ sequence. The peptides may comprise, forexample, a 5, 10, 15, 20, 25, etc residue long sequence being asubsequence of

(SEQ ID NO: 6) lealfnsvmn pkpsswrkki lpesffkepd sgshsrqsstdssgghpgpr lagg

and comprising a mutation at one or both of F52 and F53, highlighted inbold. The mutation could be to any suitable residue, such as alanine.

The peptides may be introduced into a cell, tissue, organ or individualthrough various means, such as by use of membrane translocationsequences, including for example, the whole sequence or subsequences ofthe HIV-1-trans-activating protein (Tat), Drosophila Antennapediahomeodomain protein (Antp-HD), Herpes Simplex-1 virus VP22 protein(HSV-VP22), signal-sequence-based peptides, Transportan and Amphiphilicmodel peptide, among others. These are described in detail in WO2002/007752.

TAZ and its fragments, homologues, variants and derivatives, may be madeby recombinant means. However they may also be made by synthetic meansusing techniques well known to skilled persons such as solid phasesynthesis. The proteins may also be produced as fusion proteins, forexample to aid in extraction and purification. Examples of fusionprotein partners include glutathione-S-transferase (GST), 6×His (SEQ IDNO: 4), GAL4 (DNA binding and/or transcriptional activation domains) and□-galactosidase. It may also be convenient to include a proteolyticcleavage site between the fusion protein partner and the proteinsequence of interest to allow removal of fusion protein sequences. Thefusion protein may be one which will not hinder the function of theprotein of interest sequence. Proteins may also be obtained bypurification of cell extracts from animal cells.

The TAZ polypeptides, variants, homologues, fragments and derivativesdisclosed here may be in a substantially isolated form. It will beunderstood that such polypeptides may be mixed with carriers or diluentswhich will not interfere with the intended purpose of the protein andstill be regarded as substantially isolated. A TAZ variant, homologue,fragment or derivative may also be in a substantially purified form, inwhich case it will generally comprise the protein in a preparation inwhich more than 90%, e.g. 95%, 98% or 99% of the protein in thepreparation is a protein.

The TAZ polypeptides, variants, homologues, fragments and derivativesdisclosed here may be labelled with a revealing label. The revealinglabel may be any suitable label which allows the polypeptide, etc to bedetected. Suitable labels include radioisotopes, e.g. ¹²⁵I, enzymes,antibodies, polynucleotides and linkers such as biotin. Labelledpolypeptides may be used in diagnostic procedures such as immunoassaysto determine the amount of a polypeptide in a sample. Polypeptides orlabelled polypeptides may also be used in serological or cell-mediatedimmune assays for the detection of immune reactivity to saidpolypeptides in animals and humans using standard protocols.

A TAZ polypeptides, variants, homologues, fragments and derivativesdisclosed here, optionally labelled, may also be fixed to a solid phase,for example the surface of an immunoassay well or dipstick. Suchlabelled and/or immobilised polypeptides may be packaged into kits in asuitable container along with suitable reagents, controls, instructionsand the like. Such polypeptides and kits may be used in methods ofdetection of antibodies to the polypeptides or their allelic or speciesvariants by immunoassay.

Immunoassay methods are well known in the art and will generallycomprise: (a) providing a polypeptide comprising an epitope bindable byan antibody against said protein; (b) incubating a biological samplewith said polypeptide under conditions which allow for the formation ofan antibody-antigen complex; and (c) determining whetherantibody-antigen complex comprising said polypeptide is formed.

The TAZ polypeptides, variants, homologues, fragments and derivativesdisclosed here may be used in in vitro or in vivo cell culture systemsto study the role of their corresponding genes and homologues thereof incell function, including their function in disease. For example,truncated or modified polypeptides may be introduced into a cell todisrupt the normal functions which occur in the cell. The polypeptidesmay be introduced into the cell by in situ expression of the polypeptidefrom a recombinant expression vector (see below). The expression vectoroptionally carries an inducible promoter to control the expression ofthe polypeptide.

The use of appropriate host cells, such as insect cells or mammaliancells, is expected to provide for such post-translational modifications(e.g. myristolation, glycosylation, truncation, lapidation and tyrosine,serine or threonine phosphorylation) as may be needed to confer optimalbiological activity on recombinant expression products. Such cellculture systems in which the TAZ polypeptides, variants, homologues,fragments and derivatives disclosed here are expressed may be used inassay systems to identify candidate substances which interfere with orenhance the functions of the polypeptides in the cell.

TEAD Polypeptides

TEAD1, also known as TEA domain family member 1, has a GenBank AccessionNumber of NM_(—)021961.4 or NP_(—)068780.1.

TEAD2, also known as TEA domain family member 2, has a GenBank AccessionNumber of NM_(—)003598.1 or NP_(—)003589.1.

TEAD3, also known as TEA domain family member 3, has a GenBank AccessionNumber of NM_(—)003214.3 or NP_(—)003205.2.

TEAD 4, also known as TEA domain family member 4, has a number ofisoforms. TEAD4 isoform 1 has accession number NM_(—)003213.2 orNP_(—)003204.2. TEAD4 isoform 2 has accession number NM_(—)201441.1 orNP_(—)958849.1. TEAD4 isoform 3 has accession number NM_(—)201443.1 orNP_(—)958851.1.

TAZ Nucleic Acids

The methods and compositions described here may employ, as a means fordetecting expression levels of TAZ, TAZ polynucleotides, TAZ nucleotidesand TAZ nucleic acids, as well as variants, homologues, derivatives andfragments of any of these. In addition, we disclose particular TAZfragments useful for the methods of diagnosis described here. The TAZnucleic acids may also be used for the methods of treatment orprophylaxis described.

The terms “TAZ polynucleotide”, “TAZ nucleotide” and “TAZ nucleic acid”may be used interchangeably, and should be understood to specificallyinclude both cDNA and genomic TAZ sequences. These terms are alsointended to include a nucleic acid sequence capable of encoding a TAZpolypeptide and/or a fragment, derivative, homologue or variant of this.

Where reference is made to a TAZ nucleic acid, this should be taken as areference to any member of the TAZ family of nucleic acids. Ofparticular interest are TAZ nucleic acids selected from the groupconsisting of: NM_(—)015472, NM_(—)133784, NM_(—)001037696, XM_(—)866411and NM_(—)001024869.

Also included are any one or more of the nucleic acid sequences set outas “Other TAZ nucleic acid sequences” below.

For example, the TAZ nucleic acid may comprise a human TAZ sequencehaving GenBank Accession Number NM_(—)015472.

TAZ nucleic acids may be used for a variety of means, for example,administration to an individual suffering from, or suspected to besuffering from, breast cancer, for the treatment thereof. The expressionof TAZ nucleic acids may be detected for diagnosis or detection ofcancer, in particular breast cancer. TAZ nucleic acids may also be usedfor the expression or production of TAZ polypeptides.

“Polynucleotide” generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotides” include, without limitation single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, “polynucleotide” refers to triple-stranded regions comprisingRNA or DNA or both RNA and DNA. The term polynucleotide also includesDNAs or RNAs containing one or more modified bases and DNAs or RNAs withbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications has been made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically or metabolicallymodified forms of polynucleotides as typically found in nature, as wellas the chemical forms of DNA and RNA characteristic of viruses andcells. “Polynucleotide” also embraces relatively short polynucleotides,often referred to as oligonucleotides.

It will be understood by the skilled person that numerous nucleotidesequences can encode the same polypeptide as a result of the degeneracyof the genetic code.

As used herein, the term “nucleotide sequence” refers to nucleotidesequences, oligonucleotide sequences, polynucleotide sequences andvariants, homologues, fragments and derivatives thereof (such asportions thereof). The nucleotide sequence may be DNA or RNA of genomicor synthetic or recombinant origin which may be double-stranded orsingle-stranded whether representing the sense or antisense strand orcombinations thereof. The term nucleotide sequence may be prepared byuse of recombinant DNA techniques (for example, recombinant DNA).

The term “nucleotide sequence” may means DNA.

Other Nucleic Acids

We also provide nucleic acids which are fragments, homologues, variantsor derivatives of TAZ nucleic acids. The terms “variant”, “homologue”,“derivative” or “fragment” in relation to TAZ nucleic acid include anysubstitution of, variation of, modification of, replacement of, deletionof or addition of one (or more) nucleic acids from or to the sequence ofa TAZ nucleotide sequence. Unless the context admits otherwise,references to “TAZ” and “TAZ” include references to such variants,homologues, derivatives and fragments of TAZ.

The resultant nucleotide sequence may encode a polypeptide having anyone or more TAZ activity. The term “homologue” may be intended to coveridentity with respect to structure and/or function such that theresultant nucleotide sequence encodes a polypeptide which has TAZactivity. For example, a homologue etc of TAZ may have a reducedexpression level in breast cancer cells compared to normal breast cells.With respect to sequence identity (i.e. similarity), there may be atleast 70%, at least 75%, at least 85% or at least 90% sequence identity.There may be at least 95%, such as at least 98%, sequence identity to arelevant sequence (e.g., a TAZ sequence having GenBank accession numberNM_(—)015472). These terms also encompass allelic variations of thesequences.

Variants, Derivatives and Homologues

TAZ nucleic acid variants, fragments, derivatives and homologues maycomprise DNA or RNA. They may be single-stranded or double-stranded.They may also be polynucleotides which include within them synthetic ormodified nucleotides. A number of different types of modification tooligonucleotides are known in the art. These include methylphosphonateand phosphorothioate backbones, addition of acridine or polylysinechains at the 3′ and/or 5′ ends of the molecule. For the purposes ofthis document, it is to be understood that the polynucleotides may bemodified by any method available in the art. Such modifications may becarried out in order to enhance the in vivo activity or life span ofpolynucleotides of interest.

Where the polynucleotide is double-stranded, both strands of the duplex,either individually or in combination, are encompassed by the methodsand compositions described here. Where the polynucleotide issingle-stranded, it is to be understood that the complementary sequenceof that polynucleotide is also included.

The terms “variant”, “homologue” or “derivative” in relation to anucleotide sequence include any substitution of, variation of,modification of, replacement of, deletion of or addition of one (ormore) nucleic acid from or to the sequence. Said variant, homologues orderivatives may code for a polypeptide having biological activity. Suchfragments, homologues, variants and derivatives of TAZ may comprisemodulated activity, as set out above.

As indicated above, with respect to sequence identity, a “homologue” mayhave at least 5% identity, at least 10% identity, at least 15% identity,at least 20% identity, at least 25% identity, at least 30% identity, atleast 35% identity, at least 40% identity, at least 45% identity, atleast 50% identity, at least 55% identity, at least 60% identity, atleast 65% identity, at least 70% identity, at least 75% identity, atleast 80% identity, at least 85% identity, at least 90% identity, or atleast 95% identity to the relevant sequence (e.g., a TAZ sequence havingGenBank accession number NM_(—)015472).

There may be at least 95% identity, at least 96% identity, at least 97%identity, at least 98% identity or at least 99% identity. Nucleotideidentity comparisons may be conducted as described above. A sequencecomparison program which may be used is the GCG Wisconsin Bestfitprogram described above. The default scoring matrix has a match value of10 for each identical nucleotide and −9 for each mismatch. The defaultgap creation penalty is −50 and the default gap extension penalty is −3for each nucleotide.

Hybridisation

We further describe nucleotide sequences that are capable of hybridisingselectively to any of the sequences presented herein, or any variant,fragment or derivative thereof, or to the complement of any of theabove. Nucleotide sequences may be at least 15 nucleotides in length,such as at least 20, 30, 40 or 50 nucleotides in length.

The term “hybridization” as used herein shall include “the process bywhich a strand of nucleic acid joins with a complementary strand throughbase pairing” as well as the process of amplification as carried out inpolymerase chain reaction technologies.

Polynucleotides capable of selectively hybridising to the nucleotidesequences presented herein, or to their complement, may be at least 40%homologous, at least 45% homologous, at least 50% homologous, at least55% homologous, at least 60% homologous, at least 65% homologous, atleast 70% homologous, at least 75% homologous, at least 80% homologous,at least 85% homologous, at least 90% homologous, or at least 95%homologous to the corresponding nucleotide sequences presented herein(e.g., a TAZ sequence having GenBank accession numbe NM_(—)015472). Suchpolynucleotides may be generally at least 70%, at least 80 or 90% or atleast 95% or 98% homologous to the corresponding nucleotide sequencesover a region of at least 20, such as at least 25 or 30, for instance atleast 40, 60 or 100 or more contiguous nucleotides.

The term “selectively hybridizable” means that the polynucleotide usedas a probe is used under conditions where a target polynucleotide isfound to hybridize to the probe at a level significantly abovebackground. The background hybridization may occur because of otherpolynucleotides present, for example, in the cDNA or genomic DNA librarybeing screening. In this event, background implies a level of signalgenerated by interaction between the probe and a non-specific DNA memberof the library which is less than 10 fold, such as less than 100 fold asintense as the specific interaction observed with the target DNA. Theintensity of interaction may be measured, for example, by radiolabellingthe probe, e.g. with ³²P or ³³P or with non-radioactive probes (e.g.,fluorescent dyes, biotin or digoxigenin).

Hybridization conditions are based on the melting temperature (Tm) ofthe nucleic acid binding complex, as taught in Berger and Kimmel (1987,Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152,Academic Press, San Diego Calif.), and confer a defined “stringency” asexplained below.

Maximum stringency typically occurs at about Tm-5° C. (5° C. below theTm of the probe); high stringency at about 5° C. to 10° C. below Tm;intermediate stringency at about 10° C. to 20° C. below Tm; and lowstringency at about 20° C. to 25° C. below Tm. As will be understood bythose of skill in the art, a maximum stringency hybridization can beused to identify or detect identical polynucleotide sequences while anintermediate (or low) stringency hybridization can be used to identifyor detect similar or related polynucleotide sequences.

We provide nucleotide sequences that may be able to hybridise to the TAZnucleic acids, fragments, variants, homologues or derivatives understringent conditions (e.g. 65° C. and 0.1×SSC (1×SSC=0.15 M NaCl, 0.015M Na₃ Citrate pH 7.0)).

Generation of Homologues, Variants and Derivatives

Polynucleotides which are not 100% identical to the relevant sequences(e.g., a human TAZ sequence having GenBank accession numberNM_(—)015472) but which are also included, as well as homologues,variants and derivatives of TAZ can be obtained in a number of ways.Other variants of the sequences may be obtained for example by probingDNA libraries made from a range of individuals, for example individualsfrom different populations. For example, TAZ homologues may beidentified from other individuals, or other species. Further recombinantTAZ nucleic acids and polypeptides may be produced by identifyingcorresponding positions in the homologues, and synthesising or producingthe molecule as described elsewhere in this document.

In addition, other viral/bacterial, or cellular homologues of TAZ,particularly cellular homologues found in mammalian cells (e.g. rat,mouse, bovine and primate cells), may be obtained and such homologuesand fragments thereof in general will be capable of selectivelyhybridising to human TAZ. Such homologues may be used to designnon-human TAZ nucleic acids, fragments, variants and homologues.Mutagenesis may be carried out by means known in the art to producefurther variety.

Sequences of TAZ homologues may be obtained by probing cDNA librariesmade from or genomic DNA libraries from other animal species, andprobing such libraries with probes comprising all or part of any of theTAZ nucleic acids, fragments, variants and homologues, or otherfragments of TAZ under conditions of medium to high stringency.

Similar considerations apply to obtaining species homologues and allelicvariants of the polypeptide or nucleotide sequences disclosed here.

Variants and strain/species homologues may also be obtained usingdegenerate PCR which will use primers designed to target sequenceswithin the variants and homologues encoding conserved amino acidsequences within the sequences of the TAZ nucleic acids. Conservedsequences can be predicted, for example, by aligning the amino acidsequences from several variants/homologues. Sequence alignments can beperformed using computer software known in the art. For example the GCGWisconsin PileUp program is widely used.

The primers used in degenerate PCR will contain one or more degeneratepositions and will be used at stringency conditions lower than thoseused for cloning sequences with single sequence primers against knownsequences. It will be appreciated by the skilled person that overallnucleotide homology between sequences from distantly related organismsis likely to be very low and thus in these situations degenerate PCR maybe the method of choice rather than screening libraries with labelledfragments the TAZ sequences.

In addition, homologous sequences may be identified by searchingnucleotide and/or protein databases using search algorithms such as theBLAST suite of programs.

Alternatively, such polynucleotides may be obtained by site directedmutagenesis of characterised sequences, for example, TAZ nucleic acids,or variants, homologues, derivatives or fragments thereof. This may beuseful where for example silent codon changes are required to sequencesto optimise codon preferences for a particular host cell in which thepolynucleotide sequences are being expressed. Other sequence changes maybe desired in order to introduce restriction enzyme recognition sites,or to alter the property or function of the polypeptides encoded by thepolynucleotides.

The polynucleotides described here may be used to produce a primer, e.g.a PCR primer, a primer for an alternative amplification reaction, aprobe e.g. labelled with a revealing label by conventional means usingradioactive or non-radioactive labels, or the polynucleotides may becloned into vectors. Such primers, probes and other fragments will be atleast 8, 9, 10, or 15, such as at least 20, for example at least 25, 30or 40 nucleotides in length, and are also encompassed by the term“polynucleotides” as used herein.

Polynucleotides such as a DNA polynucleotides and probes may be producedrecombinantly, synthetically, or by any means available to those ofskill in the art. They may also be cloned by standard techniques.

In general, primers will be produced by synthetic means, involving astep wise manufacture of the desired nucleic acid sequence onenucleotide at a time. Techniques for accomplishing this using automatedtechniques are readily available in the art.

Primers comprising fragments of TAZ are particularly useful in themethods of detection of TAZ expression, such as down-regulation of TAZexpression, for example, as associated with breast cancer. Suitableprimers for amplification of TAZ may be generated from any suitablestretch of TAZ. Primers which may be used include those capable ofamplifying a sequence of TAZ which is specific, i.e., does not havesignificant homology to YAP for example.

Although TAZ primers may be provided on their own, they are mostusefully provided as primer pairs, comprising a forward primer and areverse primer.

Longer polynucleotides will generally be produced using recombinantmeans, for example using a PCR (polymerase chain reaction) cloningtechniques. This will involve making a pair of primers (e.g. of about 15to 30 nucleotides), bringing the primers into contact with mRNA or cDNAobtained from an animal or human cell, performing a polymerase chainreaction under conditions which bring about amplification of the desiredregion, isolating the amplified fragment (e.g. by purifying the reactionmixture on an agarose gel) and recovering the amplified DNA. The primersmay be designed to contain suitable restriction enzyme recognition sitesso that the amplified DNA can be cloned into a suitable cloning vector

Polynucleotides or primers may carry a revealing label. Suitable labelsinclude radioisotopes such as ³²P or ³⁵S, digoxigenin, fluorescent dyes,enzyme labels, or other protein labels such as biotin. Such labels maybe added to polynucleotides or primers and may be detected using bytechniques known per se. Polynucleotides or primers or fragments thereoflabelled or unlabeled may be used by a person skilled in the art innucleic acid-based tests for detecting or sequencing polynucleotides inthe human or animal body.

Such tests for detecting generally comprise bringing a biological samplecontaining DNA or RNA into contact with a probe comprising apolynucleotide or primer under hybridising conditions and detecting anyduplex formed between the probe and nucleic acid in the sample. Suchdetection may be achieved using techniques such as PCR or byimmobilising the probe on a solid support, removing nucleic acid in thesample which is not hybridised to the probe, and then detecting nucleicacid which has hybridised to the probe. Alternatively, the samplenucleic acid may be immobilised on a solid support, and the amount ofprobe bound to such a support can be detected. Suitable assay methods ofthis and other formats can be found in for example WO89/03891 andWO90/13667.

Tests for sequencing nucleotides, for example, the TAZ nucleic acids,involve bringing a biological sample containing target DNA or RNA intocontact with a probe comprising a polynucleotide or primer underhybridising conditions and determining the sequence by, for example theSanger dideoxy chain termination method (see Sambrook et al.).

Such a method generally comprises elongating, in the presence ofsuitable reagents, the primer by synthesis of a strand complementary tothe target DNA or RNA and selectively terminating the elongationreaction at one or more of an A, C, G or T/U residue; allowing strandelongation and termination reaction to occur; separating out accordingto size the elongated products to determine the sequence of thenucleotides at which selective termination has occurred. Suitablereagents include a DNA polymerase enzyme, the deoxynucleotides dATP,dCTP, dGTP and dTTP, a buffer and ATP. Dideoxynucleotides are used forselective termination.

TAZ Control Regions

For some purposes, it may be necessary to utilise or investigate controlregions of TAZ. Such control regions include promoters, enhancers andlocus control regions. By a control region we mean a nucleic acidsequence or structure which is capable of modulating the expression of acoding sequence which is operatively linked to it.

For example, control regions are useful in generating transgenic animalsexpressing TAZ. Furthermore, control regions may be used to generateexpression constructs for TAZ. This is described in further detailbelow.

Identification of control regions of TAZ is straightforward, and may becarried out in a number of ways. For example, the coding sequence of TAZmay be obtained from an organism, by screening a cDNA library using ahuman or mouse TAZ cDNA sequence as a probe. 5′ sequences may beobtained by screening an appropriate genomic library, or by primerextension as known in the art. Database searching of genome databasesmay also be employed. Such 5′ sequences which are particularly ofinterest include non-coding regions. The 5′ regions may be examined byeye, or with the aid of computer programs, to identify sequence motifswhich indicate the presence of promoter and/or enhancer regions.

Furthermore, sequence alignments may be conducted of TAZ nucleic acidsequences from two or more organisms. By aligning TAZ sequences fromdifferent species, it is possible to determine which regions of theamino acid sequence are conserved between different species. Suchconserved regions are likely to contain control regions for the gene inquestion (i.e., TAZ). The mouse and human genomic sequences as disclosedhere, for example, a mouse TAZ genomic sequence, may be employed for,this purpose. Furthermore, TAZ homologues from other organisms may beobtained using standard methods of screening using appropriate probesgenerated from the mouse and human TAZ sequences. The genome of thepufferfish (Takifugu rubripes) or zebrafish may also be screened toidentify a TAZ homologue; thus, several zebrafish sequences of TAZ havebeen identified (noted above). Comparison of the 5′ non-coding region ofthe Fugu or zebrafish TAZ gene with a mouse or human genomic TAZsequence may be used to identify conserved regions containing controlregions.

Deletion studies may also be conducted to identify promoter and/orenhancer regions for TAZ.

The identity of putative control regions may be confirmed by molecularbiology experiments, in which the candidate sequences are linked to areporter gene and the expression of the reporter detected.

Detection and Diagnostic Methods

Detection of Expression of TAZ

We show in the Examples that the expression of TAZ in breast cancertissue is up-regulated when compared to normal breast tissue.

Accordingly, we provide for a method of diagnosis of cancer, includingbreast cancer such as metastatic, aggressive or invasive breast cancer,comprising detecting modulation of expression of TAZ, such asup-regulation of expression of TAZ in a cell or tissue of an individual.

Detection of TAZ expression, activity or amount may be used to provide amethod of determining the proliferative state of a cell. Thus, aproliferative cell is one with high levels of TAZ expression, activityor amount compared to a normal cell. Similarly, a non-proliferative cellmay be one with low levels TAZ expression, activity or amount comparedto a normal cell.

Such detection may also be used to determine whether a cell will becomeinvasive or aggressive. Thus, detection of a high level of TAZexpression, amount or activity of TAZ in the cell may indicate that thecell is likely to be or become aggressive, metastatic or invasive.Similarly, if a cell has a low level of TAZ expression, amount oractivity, the cell is not or is not likely to be aggressive, metastaticor invasive.

It will be appreciated that as the level of TAZ varies with theaggressiveness of a tumour, that detection of TAZ expression, amount oractivity may also be used to predict a survival rate of an individualwith cancer, i.e., high levels of TAZ indicating a lower survival rateor probability and low levels of TAZ indicating a higher survival rateor probability, both as compared to individuals or cognate populationswith normal levels of TAZ. Detection of expression, amount or activityof TAZ may therefore be used as a method of prognosis of an individualwith cancer.

Detection of TAZ expression, amount or level may be used to determinethe likelihood of success of a particular therapy in an individual witha cancer. It may be used in a method of determining whether a tumour inan individual is, or is likely to be, an invasive or metastatic tumour.

The diagnostic methods described in this document may be combined withthe therapeutic methods described. Thus, we provide for a method oftreatment, prophylaxis or alleviation of cancer in an individual, themethod comprising detecting modulation of expression, amount or activityof TAZ in a cell of the individual and administering an appropriatetherapy to the individual based on the aggressiveness of the tumour.

Typically, physical examination of the breast and X-ray mammography isused for the detection of breast cancer. A biopsy of the tumour istypically taken for histopathological examination for the diagnosis ofbreast cancer. Detection of TAZ expression, amount or activity can beused to diagnose, or further confirm the diagnosis of, breast cancer,along with the standard histopathological procedures. This may beespecially useful when the histopathological analysis does not yield aclear result.

The presence and quantity of TAZ polypeptides and nucleic acids may bedetected in a sample as described in further detail below. Thus, the TAZassociated diseases, including breast cancer, can be diagnosed bymethods comprising determining from a sample derived from a subject anabnormally decreased or increased expression, amount or activity, suchas a increased expression, amount or activity, of the TAZ polypeptide orTAZ mRNA.

The sample may comprise a cell or tissue sample from an organism orindividual suffering or suspected to be suffering from a diseaseassociated with increased, reduced or otherwise abnormal TAZ expression,amount or activity, including spatial or temporal changes in level orpattern of expression, amount or activity. The level or pattern ofexpression, amount or activity of TAZ in an organism suffering from orsuspected to be suffering from such a disease may be usefully comparedwith the level or pattern of expression, amount or activity in a normalorganism as a means of diagnosis of disease.

The sample may comprise a cell or tissue sample from an individualsuffering or suspected to be suffering from breast cancer, such as abreast tissue or cell sample.

In some embodiments, an increased level of expression, amount oractivity of TAZ is detected in the sample. The level of TAZ may beincreased to a significant extent when compared to normal cells, orcells known not to be cancerous. Such cells may be obtained from theindividual being tested, or another individual, such as those matched tothe tested individual by age, weight, lifestyle, etc.

In some embodiments, the level of expression, amount or activity of TAZis increased by 10%, 20%, 30% or 40% or more. In some embodiments, thelevel of expression, amount or activity of TAZ is increased by 45% ormore, such as 50% or more, as judged by cDNA hybridisation.

The expression, amount or activity of TAZ may be detected in a number ofways, as known in the art, and as described in further detail below.Typically, the amount of TAZ in a sample of tissue from an individual ismeasured, and compared with a sample from an unaffected individual. BothTAZ nucleic acid, as well as TAZ polypeptide levels may be measured.

Detection of the amount, activity or expression of TAZ may be used tograde breast cancer. For example, a high level of amount, activity orexpression of TAZ may indicate an aggressive, invasive or metastaticcancer. Similarly, a low level of amount, activity or expression of TAZmay indicate a non-aggressive, non-invasive or non-metastatic cancer.Such a grading system may be used in conjunction with establishedgrading systems such as the Elston-Ellis modified Scarff, Bloom,Richardson grading system, also known as the Nottingham grading system(NGS) (5, 6, Haybittle et al, 1982).

This system is the most studied and widely used method of breast tumorgrading. The NGS is based on a phenotypic scoring procedure thatinvolves the microscopic evaluation of morphologic and cytologicfeatures of tumor cells including degree of tubule formation, nuclearpleomorphism and mitotic count (6). The sum of these scores stratifiesbreast tumors into grade I (G1) (well-differentiated, slow-growing),grade II (G2) (moderately differentiated), and grade III (G3)(poorly-differentiated, highly-proliferative) malignancies.

Levels of TAZ gene expression may be determined using a number ofdifferent techniques.

Measuring Expression of TAZ at the RNA Level

TAZ gene expression can be detected at the RNA level.

In one embodiment therefore, we disclose a method of detecting thepresence of a nucleic acid comprising a TAZ nucleic acid in a sample, bycontacting the sample with at least one nucleic acid probe which isspecific for the TAZ nucleic acid and monitoring said sample for thepresence of the TAZ nucleic acid. For example, the nucleic acid probemay specifically bind to the TAZ nucleic acid, or a portion of it, andbinding between the two detected; the presence of the complex itself mayalso be detected.

Thus, in one embodiment, the amount of TAZ nucleic acid in the form ofTAZ mRNA may be measured in a sample. TAZ mRNA may be assayed by in situhybridization, Northern blotting and reverse transcriptase—polymerasechain reaction. Nucleic acid sequences may be identified by in situhybridization, Southern blotting, single strand conformationalpolymorphism, PCR amplification and DNA-chip analysis using specificprimers. (Kawasaki, 1990; Sambrook, 1992; Lichter et al, 1990; Orita etal, 1989; Fodor et al., 1993; Pease et al., 1994).

TAZ RNA may be extracted from cells using RNA extraction techniquesincluding, for example, using acid phenol/guanidine isothiocyanateextraction (RNAzol B; Biogenesis), or RNeasy RNA preparation kits(Qiagen). Typical assay formats utilising ribonucleic acid hybridisationinclude nuclear run-on assays, RT-PCR and RNase protection assays(Melton et al., Nuc. Acids Res. 12:7035. Methods for detection which canbe employed include radioactive labels, enzyme labels, chemiluminescentlabels, fluorescent labels and other suitable labels.

Each of these methods allows quantitative determinations to be made, andare well known in the art. Decreased or increased TAZ expression, amountor activity can therefore be measured at the RNA level using any of themethods well known in the art for the quantitation of polynucleotides.Any suitable probe from a TAZ sequence, for example, any portion of asuitable human TAZ sequence may be used as a probe. Sequences fordesigning TAZ probes may include a sequence having accession numberNM_(—)015472, or a portion thereof.

Typically, RT-PCR is used to amplify RNA targets. In this process, thereverse transcriptase enzyme is used to convert RNA to complementary DNA(cDNA) which can then be amplified to facilitate detection.

Many DNA amplification methods are known, most of which rely on anenzymatic chain reaction (such as a polymerase chain reaction, a ligasechain reaction, or a self-sustained sequence replication) or from thereplication of all or part of the vector into which it has been cloned.

Many target and signal amplification methods have been described in theliterature, for example, general reviews of these methods in Landegren,U. et al., Science 242:229-237 (1988) and Lewis, R., Genetic EngineeringNews 10:1, 54-55 (1990).

For example, the polymerase chain reaction may be employed to detect TAZmRNA.

The “polymerase chain reaction” or “PCR” is a nucleic acid amplificationmethod described inter alia in U.S. Pat. Nos. 4,683,195 and 4,683,202.PCR can be used to amplify any known nucleic acid in a diagnosticcontext (Mok et al., 1994, Gynaecologic Oncology 52:247-252).Self-sustained sequence replication (3SR) is a variation of TAS, whichinvolves the isothermal amplification of a nucleic acid template viasequential rounds of reverse transcriptase (RT), polymerase and nucleaseactivities that are mediated by an enzyme cocktail and appropriateoligonucleotide primers (Guatelli et al., 1990, Proc. Natl. Acad. Sci.USA 87:1874). Ligation amplification reaction or ligation amplificationsystem uses DNA ligase and four oligonucleotides, two per target strand.This technique is described by Wu, D. Y. and Wallace, R. B., 1989,Genomics 4:560. In the Qβ Replicase technique, RNA replicase for thebacteriophage Qβ, which replicates single-stranded RNA, is used toamplify the target DNA, as described by Lizardi et al., 1988,Bio/Technology 6:1197.

A PCR procedure basically involves: (1) treating extracted DNA to formsingle-stranded complementary strands; (2) adding a pair ofoligonucleotide primers, wherein one primer of the pair is substantiallycomplementary to part of the sequence in the sense strand and the otherprimer of each pair is substantially complementary to a different partof the same sequence in the complementary antisense strand; (3)annealing the paired primers to the complementary sequence; (4)simultaneously extending the annealed primers from a 3′ terminus of eachprimer to synthesize an extension product complementary to the strandsannealed to each primer wherein said extension products after separationfrom the complement serve as templates for the synthesis of an extensionproduct for the other primer of each pair; (5) separating said extensionproducts from said templates to produce single-stranded molecules; and(6) amplifying said single-stranded molecules by repeating at least oncesaid annealing, extending and separating steps.

Reverse transcription-polymerase chain reaction (RT-PCR) may beemployed. Quantitative RT-PCR may also be used. Such PCR techniques arewell known in the art, and may employ any suitable primer from a TAZsequence.

Alternative amplification technology can also be exploited. For example,rolling circle amplification (Lizardi et al., 1998, Nat Genet 19:225) isan amplification technology available commercially (RCAT™) which isdriven by DNA polymerase and can replicate circular oligonucleotideprobes with either linear or geometric kinetics under isothermalconditions. A further technique, strand displacement amplification (SDA;Walker et al., 1992, Proc. Natl. Acad. Sci. USA 80:392) begins with aspecifically defined sequence unique to a specific target.

Measuring Expression of TAZ at the Polypeptide Level

TAZ expression can be detected at the polypeptide level.

In a further embodiment, therefore, TAZ expression, amount or activitymay be detected by detecting the presence or amount of TAZ polypeptidein a sample. This may be achieved by using molecules which bind to TAZpolypeptide. Suitable molecules/agents which bind either directly orindirectly to the TAZ polypeptide in order to detect its presenceinclude naturally occurring molecules such as peptides and proteins, forexample antibodies, or they may be synthetic molecules.

Thus, we disclose a method of detecting the presence of a TAZpolypeptide by contacting a cell sample with an antibody capable ofbinding the polypeptide and monitoring said sample for the presence ofthe polypeptide.

For example, the TAZ polypeptide may be detected using an anti-TAZantibody. Such antibodies may be made by means known in the art (asdescribed in further detail below). For example, an anti-TAZ antibodymay comprise an antibody to TAZ amino acid residues 160-229 of TAZ,e.g., an anti-peptide antibody.

This may conveniently be achieved by monitoring the presence of acomplex formed between the antibody and the polypeptide, or monitoringthe binding between the polypeptide and the antibody. Methods ofdetecting binding between two entities are known in the art, and includeFRET (fluorescence resonance energy transfer), surface plasmonresonance, etc.

Standard laboratory techniques such as immunoblotting as described abovecan be used to detect altered levels of TAZ protein, as compared withuntreated cells in the same cell population.

Gene expression may also be determined by detecting changes inpost-translational processing of TAZ polypeptides orpost-transcriptional modification of TAZ nucleic acids. For example,differential phosphorylation of TAZ polypeptides, the cleavage of TAZpolypeptides or alternative splicing of TAZ RNA, and the like may bemeasured. Levels of expression of gene products such as TAZpolypeptides, as well as their post-translational modification, may bedetected using proprietary protein assays or techniques such as 2Dpolyacrylamide gel electrophoresis.

Assay techniques that can be used to determine levels of TAZ protein ina sample derived from a host are well-known to those of skill in theart. Antibodies can be assayed for immunospecific binding by any methodknown in the art.

The immunoassays which can be used include but are not limited tocompetitive and non-competitive assay systems using techniques such aswestern blots, radioimmunoassays, ELISA, sandwich immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays and protein A immunoassays. Such assays are routine in theart (see, for example, Ausubel et al., eds, 1994, Current Protocols inMolecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which isincorporated by reference herein in its entirety).

The specimen may be assayed for polypeptides/proteins byimmunohistochemical and immunocytochemical staining (see generallyStites and Terr, Basic and Clinical Immunology, Appleton and Lange,1994), ELISA, RIA, immunoblots, Western blotting, immunoprecipitation,functional assays and protein truncation test. Other assay methodsinclude radioimmunoassays, competitive-binding assays, Western Blotanalysis and ELISA assays.

ELISA assays are well known to those skilled in the art. Both polyclonaland monoclonal antibodies may be used in the assays. Where appropriateother immunoassays, such as radioimmunoassays (RIA) may be used as areknown to those in the art. Available immunoassays are extensivelydescribed in the patent and scientific literature. See, for example,U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987;3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345;4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521 as well asSambrook et al, 1992.

Detecting Expression of TAZ-Induced Polypeptides

As shown in the Examples, TAZ induces the expression of IGFBP3, ADAMTS1,CTGF, Cyr61, FSTL1, FN1, FBN1, FBN2AXL, ITGB2, CRIM1 and Alcam.

IFGBP3 has a GenBank Accession number AAA52706. ADAMTS1 has a GenBankAccession number AAZ73034. CTGF has a GenBank Accession number CAA63267.Cyr61 has a GenBank Accession number CAG38757. FSTL1 has a GenBankAccession number NP_(—)009016. FN1 has a GenBank Accession numberAAH05858. FBN1 has a GenBank Accession number AAH94721. ITGB2 has aGenBank Accession number AAH21077. CRIM1 has a GenBank Accession numberAAQ88737. Alcam has a GenBank Accession number AAB59499.

Accordingly, TAZ expression may be detected using the expression of anyone or more of these proteins as proxy, at either the nucleic acid levelor the polypeptide level, as described above.

More generally, the expression of any one or more of these proteins maybe detected as a means of diagnosis of cancer, including breast cancersuch as metastatic, aggressive or invasive breast cancer, or anoncogenic or metastatic cell. Detection of such expression may be doneby any of the methods described above.

Diagnostic Kits

We also provide diagnostic kits for detecting breast cancer in anindividual, or susceptibility to breast cancer in an individual.

The diagnostic kit may comprise means for detecting expression, amountor activity of TAZ in the individual, by any means as described in thisdocument. The diagnostic kit may therefore comprise any one or more ofthe following: a TAZ polynucleotide or a fragment thereof; acomplementary nucleotide sequence to TAZ nucleic acid or a fragmentthereof; a TAZ polypeptide or a fragment thereof, or an antibody to aTAZ, such as comprising an anti-TAZ antibody against amino acid residues160-229 of TAZ, e.g., an anti-peptide antibody human TAZ antibody.

The diagnostic kit may comprise instructions for use, or other indicia.The diagnostic kit may further comprise means for treatment orprophylaxis of breast cancer, such as any of the compositions describedin this document, or any means known in the art for treating breastcancer. In particular, the diagnostic kit may comprise an anti-TAZ agentas described, for example obtained by screening. The diagnostic kit maycomprise a therapeutic drug such as Tamoxifen (Nolvadex) or its variantssuch as tamoxifen, tamoxifen citrate or any other antiestrogen orestrogen blocker. The therapeutic drug may also comprise an anti-TAZantibody.

Prophylactic and Therapeutic Methods

We disclose methods of treating an abnormal conditions, such as breastcancer, related to insufficient amounts of TAZ expression or activity.Methods of preventing breast cancer (i.e., prophylaxis) also suitablyemploy the same or similar approaches.

In general terms, our methods involve manipulation of cancer cells, bymodulating (such as down-regulating) the expression, amount or activityof TAZ in the cell. A step of detecting modulated TAZ expression, amountor activity in a cell may be conducted before or after the manipulationstep. The detection step may detect up-regulated or down-regulated TAZexpression, amount or activity. Any of the methods of modulating ordown-regulating TAZ, as described in detail elsewhere in this document,may be used.

The method may comprise exposing the cell to an siRNA or shRNA or ananti-TAZ antibody capable of specifically binding to TAZ. TAZ may bemodulated by targeting a TAZ target site selected from KD-1(5′-GATGAATCCGGCCTCGGCGCC-3′ (SEQ ID NO: 1)), KD-650(5′-AGAGGTACTTCCTCAATCA-3′ (SEQ ID NO: 2)) or KD-652(5′-AGGTACTTCCTCAATCACA-3′ (SEQ ID NO: 3)).

According to our methods, the cancer cell becomes non-cancerous or theinvasive or metastatic cancer cell becomes non-invasive ornon-metastatic as a result of the manipulation. The cancer may inparticular comprise breast cancer. It may comprise invasive ormetastatic cancer such as Invasive Ductal Carcinoma (IDC).

As TAZ is associated with aggressiveness and invasiveness of cancer, thelevel of TAZ may be detected in a cell of an individual with cancer, ina cancer or non-cancer cell, and the aggressiveness of the cancerassessed. A high level of TAZ amount, expression or activity comparedwith a normal cell indicates an aggressive or invasive cancer, and astronger or harsher therapy may therefore be required and chosen.Similarly, a lower level may indicate a less aggressive or invasivetherapy.

The approaches described here may be used for therapy of any TAZ relateddisease in general. TAZ related diseases include proliferative diseasesand in particular include cancer. For example, a TAZ related disease mayinclude breast cancer, such as metastatic, invasive or aggressive breastcancer.

A TAZ related disease is defined as being “treated” if a conditionassociated with the disease is significantly inhibited (i.e., by 50% ormore) relative to controls. The inhibition may be by at least 75%relative to controls, such as by 90%, by 95% or 100% relative tocontrols. The condition may comprise cell proliferation, or it maycomprise cell cycle time, cell number, cell migration, cellinvasiveness, etc. By the term “treatment” we mean to also includeprophylaxis or alleviation of cancer.

TAZ polypeptide represents a target for inhibition of its function fortherapy, particularly in tumour cells and other proliferative cells.

The term proliferative disorder is used herein in a broad sense toinclude any disorder that requires control of the cell cycle. Inparticular, a proliferative disorder includes malignant andpre-neoplastic disorders. The methods and compositions described hereare especially useful in relation to treatment or diagnosis ofadenocarcinomas such as: small cell lung cancer, and cancer of thekidney, uterus, prostrate, bladder, ovary, colon and breast. Forexample, malignancies which may be treatable include acute and chronicleukemias, lymphomas, myelomas, sarcomas such as Fibrosarcoma,myxosarcoma, liposarcoma, lymphangioendotheliosarcoma, angiosarcoma,endotheliosarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,lymphangiosarcoma, synovioma, mesothelioma, leimyosarcoma,rhabdomyosarcoma, colon carcinoma, ovarian cancer, glioma, prostatecancer, pancreatic cancer, breast cancer, squamous cell carcinoma, basalcell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma,choriocarcinoma, renal cell carcinoma, hepatoma, bile duct carcinomaseminoma, embryonal carcinoma, cervical cancer, testicular tumour, lungcarcinoma, small cell lung carcinoma, bladder carcinoma, epithelialcarcinoma, glioma, astrocytoma, ependymoma, pinealoma, hemangioblastoma,acoustic neuoma, medulloblastoma, craniopharyngioma, oligodendroglioma,menangioma, melanoma, neutroblastoma and retinoblastoma.

One possible approach for therapy of such disorders is to expressanti-sense constructs directed against TAZ polynucleotides as describedhere, and administering them to tumour cells, to inhibit gene functionand prevent the tumour cell from growing or progressing.

Anti-sense constructs may be used to inhibit gene function to preventgrowth or progression in a proliferative cell. Antisense constructs,i.e., nucleic acid, such as RNA, constructs complementary to the sensenucleic acid or mRNA, are described in detail in U.S. Pat. No. 6,100,090(Monia et al.), and Neckers et al., 1992, Crit Rev Oncog 3(1-2):175-231,the teachings of which document are specifically incorporated byreference.

In a particular example, breast cancer may be treated or prevented byreducing the amount, expression or activity of TAZ in whole or in part,for example by siRNAs capable of binding to and destroying TAZ mRNA. Wespecifically provide for an anti-TAZ agent which downregulates TAZ byRNA interference. The anti-TAZ agent may comprise a Small InterferingRNA (siRNA) or Short Hairpin RNA (shRNA).

The anti-TAZ agent may comprise shRNA 1 (sense oligonucleotide sequence5′-GATGAATCCGGCCTCGGCGCC-3′ (SEQ ID NO: 1)), shRNA 650 (senseoligonucleotide sequence 5′-AGAGGTACTTCCTCAATCA-3′ (SEQ ID NO: 2)) orshRNA 652 (sense oligonucleotide sequence 5′-AGGTACTTCCTCAATCACA-3′ (SEQID NO: 3)). Methods of producing such shRNAs are described below and indetail in the Examples.

RNA interference (RNAi) is a method of post transcriptional genesilencing (PTGS) induced by the direct introduction of double-strandedRNA (dsRNA) and has emerged as a useful tool to knock out expression ofspecific genes in a variety of organisms. RNAi is described by Fire etal., Nature 391:806-811 (1998). Other methods of PTGS are known andinclude, for example, introduction of a transgene or virus. Generally,in PTGS, the transcript of the silenced gene is synthesised but does notaccumulate because it is rapidly degraded. Methods for PTGS, includingRNAi are described, for example, in the Ambion.com world wide web site,in the directory “/hottopics/”, in the “rnai” file.

Suitable methods for RNAi in vitro are described herein. One such methodinvolves the introduction of siRNA (small interfering RNA). Currentmodels indicate that these 21-23 nucleotide dsRNAs can induce PTGS.Methods for designing effective siRNAs are described, for example, inthe Ambion web site described above. RNA precursors such as ShortHairpin RNAs (shRNAs) can also be encoded by all or a part of the TAZnucleic acid sequence.

Alternatively, double-stranded (ds) RNA is a powerful way of interferingwith gene expression in a range of organisms that has recently beenshown to be successful in mammals (Wianny and Zernicka-Goetz, 2000, NatCell Biol 2:70-75). Double stranded RNA corresponding to the sequence ofa TAZ polynucleotide can be introduced into or expressed in oocytes andcells of a candidate organism to interfere with TAZ activity.

Other methods of modulating TAZ gene expression are known to thoseskilled in the art and include dominant negative approaches. Thus,another approach is to use non-functional variants of TAZ polypeptide inthis document that compete with the endogenous gene product resulting ininhibition of function.

TAZ gene expression may also be modulated by as introducing peptides orsmall molecules which inhibit gene expression or functional activity.Thus, compounds identified by the assays described here as binding to ormodulating, such as down-regulating, the amount, activity or expressionof TAZ polypeptide may be administered to tumour or proliferative cellsto prevent the function of TAZ polypeptide. Such a compound may beadministered along with a pharmaceutically acceptable carrier in anamount effective to down-regulate expression or activity TAZ, or byactivating or down-regulating a second signal which controls TAZexpression, activity or amount, and thereby alleviating the abnormalcondition.

Suitable antibodies against TAZ polypeptide as described herein may alsobe used as therapeutic agents. An anti-TAZ antibody may comprise arabbit anti-TAZ antibody against amino acids 160-229 of TAZ.Furthermore, the anti-TAZ antibody may comprise any one or more of thefollowing: a rabbit anti-TAZ antibody (catalogue number 2149S, CellSignaling Technology, Danvers, Mass., USA), rabbit polyclonal anti-TAZantibody (catalogue number NB110-58359SS, Novus Biological, Littleton,Colo., USA), Mouse Monoclonal anti-TAZ [1B10] (catalogue numberH00006901-M12, Novus Biological, Littleton, Colo., USA), Rabbitanti-Human TAZ Polyclonal Antibody (catalogue number LS-B94, LifeSpanBiosciences, Inc., Seattle, Wash., USA) or p-TAZ (Ser 89)-R (cataloguenumber sc-17610-R, Santa Cruz Biotechnology, Santa Cruz, Calif., USA).

Alternatively, gene therapy may be employed to control the endogenousproduction of TAZ by the relevant cells such as breast cells in thesubject. For example, a polynucleotide encoding a TAZ siRNA or a portionof this may be engineered for expression in a replication defectiveretroviral vector, as discussed below. The retroviral expressionconstruct may then be isolated and introduced into a packaging celltransduced with a retroviral plasmid vector containing RNA encoding ananti-TAZ siRNA such that the packaging cell now produces infectiousviral particles containing the sequence of interest. These producercells may be administered to a subject for engineering cells in vivo andregulating expression of the TAZ polypeptide in vivo. For overview ofgene therapy, see Chapter 20, Gene Therapy and other MolecularGenetic-based Therapeutic Approaches, (and references cited therein) inHuman Molecular Genetics, T Strachan and A P Read, BIOS ScientificPublishers Ltd (1996).

In some embodiments, the level of TAZ is decreased in a breast cell.Furthermore, in such embodiments, treatment may be targeted to, orspecific to, breast cells. The expression of TAZ may be specificallydecreased only in diseased breast cells (i.e., those cells which arecancerous), and not substantially in other non-diseased breast cells. Inthese methods, expression of TAZ may be not substantially reduced inother cells, i.e., cells which are not breast cells. Thus, in suchembodiments, the level of TAZ remains substantially the same or similarin non-breast cells in the course of or following treatment.

Breast cell specific reduction of TAZ levels may be achieved by targetedadministration, i.e., applying the treatment only to the breast cellsand not other cells. However, in other embodiments, down-regulation ofTAZ expression in breast cells (and not substantially in other cell ortissue types) is employed. Such methods may advantageously make use ofbreast specific expression vectors, for breast specific expression offor example siRNAs, as described in further detail below.

Breast-Specific Expression of a Transgene (Anti-TAZ siRNA)

Cancer gene therapy has to selectively target tumour tissues so as toreduce undesired side effects in normal tissue. Targeting transgeneexpression to malignant tissues requires the use of specific regulatoryelements including promoters based on tumour biology, tissue-specificpromoters and inducible regulatory elements (A 1).

Promoters Based on Tumour Biology

Certain genes are upregulated in breast cancer. The promoters of thesegenes can be used to drive tumour-selective expression of a transgeneusing a recombinant replication-defective retroviral vectors. Examplesof such genes include the vascular endothelial growth factor (VEGF),vascular endothelial growth factor receptor-1 (VEGFR-1) and VEGFR-2,which are known to be upregulated in breast cancer in a tumour-stagedependent manner (A2). c-erbB2 oncogene is selectively upregulated inbreast carcinomas (A3, A6). L-plastin, a human actin-binding protein isconstitutively and abundantly expressed in malignant epithelial cellsbut not in normal tissue, except for low-level expression in maturehematopoietic cells (A4). Anti-apoptotic gene Bcl-2 has been found to beupregulated in breast cancer cells (A5). Human breast tumours expresshigh levels of MUC1 compared to normal breast tissues (A7).

Tissue Specific Promoters

Certain genes are expressed specifically in breast tissues. Examples ofsuch genes are the human α-lactalbumin (ALA) and ovine β-lactoglobulin(BLG). The promoters of such genes can be used to drive the expressionof transgenes in adenoviral vectors in a breast cancer cell-specificmanner (A8). Gene therapy for breast carcinoma may be approached bytailoring a virus with affinity to this tissue, such as the mousemammary tumour virus (MMTV). The glucorticoid-responsive long terminalrepeats (LTR) of this retrovirus can be used as promoter forglucocorticoid-induced the expression of a transgene (A9).

Inducible Promoters

Inducible promoters are used as mediators of transient transgeneexpression. Various stress genes are upregulated in breast tumours uponirradiation or chemotherapeutic treatment. Examples of such stress genesare heat shock protein (HSP) (A10) and multidrug resistance gene-1(MDR-1) (A11). The promoters of these genes can therefore be used todrive the tumour specific expression of a transgene in breast cancersthat have been subjected to irradiation or chemotherapy.

Transcriptionally targeted gene therapy is usually achieved by directintratumour injection of a replication-defective adenoviral expressionvector containing the transgene of interest (A6, A12, A13). Thetransgene can also be delivered by intratumoural injection as a lipidcomplex with cationic liposomes (A14, A15).

Breast Cancer

According to the methods and compositions described here, TAZ is usefulfor diagnosing or treating breast cancer. Where this document refers to“cancer”, this should be taken to include metastatic, aggressive orinvasive cancer.

There are several types of breast cancer. The most common is ductalcarcinoma, which begins in the lining of the milk ducts of the breast.Another type, lobular carcinoma, begins in the lobules where breast milkis produced. If a malignant tumor invades nearby tissue, it is known asinfiltrating or invasive cancer. When breast cancer spreads outside thebreast, cancer cells often are found in the lymph nodes under the arm.Breast cancer cells may spread beyond the breast such as to other lymphnodes, the bones, liver, or lungs.

The recognised stages of breast cancer comprise:

Stage 0: Very early breast cancer. This type of cancer has not spreadwithin or outside the breast. It is sometimes called DCIS, LCIS, orbreast cancer in situ or non-invasive cancer.

Stage I: The cancer is no larger than about 1 inch in size and has notspread outside the breast. (also described as early breast cancer.)

Stage II: The presence of any of the following: the cancer is no largerthan 1 inch, but has spread to the lymph nodes under the arm; the canceris between 1 and 2 inches. It may or may not have spread to the lymphnodes under the arm; the cancer is larger than 2 inches, but has notspread to the lymph nodes under the arm.

Stage III and Stage IIIA: The presence of any of the following: thecancer is smaller than 2 inches and has spread to the lymph nodes underthe arm, the cancer also is spreading further to other lymph nodes; thecancer is larger than 2 inches and has spread to the lymph nodes underthe arm.

Stage IIIB: The presence of any of the following: the cancer has spreadto tissues near the breast (skin, chest wall, including the ribs and themuscles in the chest); the cancer has spread to lymph nodes inside thechest wall along the breast bone.

Stage IV: The cancer has spread to other parts of the body, most oftenthe bones, lungs, liver, or brain. Or, the tumor has spread locally tothe skin and lymph nodes inside the neck, near the collarbone.

Inflammatory Breast Cancer: Inflammatory breast cancer is a rare, butvery serious, aggressive type of breast cancer. The breast may look redand feel warm. There may be ridges, welts, or hives on the breast; orthe skin may look wrinkled. It is sometimes misdiagnosed as a simpleinfection.

Recurrent Breast Cancer: Recurrent disease means that the cancer hascome back (recurred) after it has been treated. It may come back in thebreast, in the soft tissues of the chest (the chest wall), or in anotherpart of the body.

Breast Cancer In Situ—DCIS and LCIS

Many breast cancers being found are very early cancers known as breastcancer in situ or noninvasive cancer. Most of these cancers are found bymammography. These very early cell changes may become invasive breastcancer. Two types of breast cancer in situ include the following:

DCIS (ductal carcinoma in situ), which means that abnormal cells arefound only in the lining of a milk duct of the breast. The abnormalcells have not spread outside the duct. They have not spread within thebreast, beyond the breast, to the lymph nodes under the arm, or to otherparts of the body. There are several types of DCIS. If not removed, sometypes may change over time and become invasive cancers. Some may neverbecome invasive cancers. (DCIS is sometimes called intraductalcarcinoma.)

LCIS (lobular carcinoma in situ), which means that abnormal cells arefound in the lining of a milk lobule. Although LCIS is not considered tobe actual breast cancer at this noninvasive stage, it is a warning signof increased risk of developing invasive cancer. LCIS is sometimes foundwhen a biopsy is done for another lump or unusual change that is foundon a mammogram. Patients with LCIS have a 25 percent chance ofdeveloping breast cancer in either breast during the next 25 years.

Microcalcifications are very small specks of calcium that can't be felt,but can be seen on a mammogram. They are formed by rapidly dividingcells. When they are clustered in one area of the breast, this could bean early sign of breast cancer in situ. About half of the breast cancersfound by mammography appear as clusters of microcalcifications. Theother half appear as lumps.

Diagnosis

Our diagnostic methods may be used in conjunction with any known methodof diagnosis of breast cancer, including detecting of mutations ineither or both of the known breast cancer genes BRCA1 and BRCA2.Alternatively, or in addition, the diagnosis may be carried out bydetection of Her2 expression, for example by use of anti-Her2 antibody.

Treatment

Known treatments for breast cancer may consist of any one or more of thefollowing: Surgery, radiation therapy, chemotherapy, high-dosechemotherapy, hormonal therapy and immunotherapy. Accordingly, any ofthe treatment methods described here may be combined with any one ormore of the preceding known therapies. In addition, any one or more ofthe following general therapies known to be effective for treatment oralleviation of cancer may be used.

Nonspecific Immunomodulating Agents

Nonspecific immunomodulating agents are substances that stimulate orindirectly augment the immune system. Often, these agents target keyimmune system cells and cause secondary responses such as increasedproduction of cytokines and immunoglobulins. Two nonspecificimmunomodulating agents used in cancer treatment are bacillusCalmette-Guerin (BCG) and levamisole. The anti-TAZ agents described heremay be used in conjunction with any of such nonspecific immunomodulatingagents.

Biological Response Modifiers

Some antibodies, cytokines, and other immune system substances can beproduced in the laboratory for use in cancer treatment. These substancesare often called biological response modifiers (BRMs). They alter theinteraction between the body's immune defenses and cancer cells toboost, direct, or restore the body's ability to fight the disease. BRMsinclude interferons, interleukins, colony-stimulating factors,monoclonal antibodies, and vaccines. The anti-TAZ agents described heremay be used in conjunction with any of such biological responsemodifiers.

Interferons (IFN)

There are three major types of interferons—interferon alpha, interferonbeta, and interferon gamma; interferon alpha is the type most widelyused in cancer treatment.

Interferons can improve the way a cancer patient's immune system actsagainst cancer cells. In addition, interferons may act directly oncancer cells by slowing their growth or promoting their development intocells with more normal behavior. Some interferons may also stimulate NKcells, T cells, and macrophages, boosting the immune system's anticancerfunction.

The anti-TAZ agents described here may be used in conjunction with anyof such interferons.

Interleukins (IL)

Like interferons, interleukins are cytokines that occur naturally in thebody. Many interleukins have been identified; interleukin-2 (IL-2 oraldesleukin) has been the most widely studied in cancer treatment. IL-2stimulates the growth and activity of many immune cells, such aslymphocytes, that can destroy cancer cells.

The anti-TAZ agents described here may be used in conjunction with anyof such interleukins.

Colony-Stimulating Factors (CSFs)

Colony-stimulating factors (CSFs) (sometimes called hematopoietic growthfactors) usually do not directly affect tumor cells; rather, theyencourage bone marrow stem cells to divide and develop into white bloodcells, platelets, and red blood cells. Bone marrow is critical to thebody's immune system because it is the source of all blood cells.

G-CSF (filgrastim) and GM-CSF (sargramostim) can increase the number ofwhite blood cells, thereby reducing the risk of infection in patientsreceiving chemotherapy. G-CSF and GM-CSF can also stimulate theproduction of stem cells in preparation for stem cell or bone marrowtransplants; Erythropoietin can increase the number of red blood cellsand reduce the need for red blood cell transfusions in patientsreceiving chemotherapy; and Oprelvekin can reduce the need for platelettransfusions in patients receiving chemotherapy.

The anti-TAZ agents described here may be used in conjunction with anyof such colony-stimulating factors.

Monoclonal Antibodies (MOABs)

Herceptin is used to treat metastatic breast cancer in patients withtumors that produce excess amounts of a protein called HER-2.(Approximately 25 percent of breast cancer tumors produce excess amountsof HER-2). In particular embodiments, the methods of treatment describedhere may be used in combination with administration of anti-Her2antibody, for example, Herceptin, to the individual concerned.

The anti-TAZ agents described here may be used in conjunction with anyof such monoclonal antibodies.

Her2/Neu

The HER-2/neu (erbB-2) gene product is a 185-kDA transmembrane receptortyrosine kinase that belongs to the family of receptors for epidermalgrowth factor. It is described in some detail in Reese, D. M., et al.,Stem Cells, 15, 1-8 (1997) which is incorporated herein by reference.

Recently, enormous attention has been given to the importance ofHER-2/neu in breast cancer. HER-2/neu is overexpressed in 20-30% ofhuman breast cancers and the increased expression has been associatedwith poor prognosis. The discovery of this has led to the development ofHERCEPTIN, an antibody to HER-2/neu, which in tests has been found tolengthen remission time in metastatic breast cancer. HER-2/neu is acell-surface receptor that transmits growth signals to the cell nucleus.HERCEPTIN appears to block these signals thereby apparently inhibitingproliferation of cells mediated by HER-2/neu in HER-2/neu positivebreast cancer.

Overexpression of HER-2/neu has also been found in a portion of ovariancancers, gastric cancers, endometrial cancers, salivary cancers,pancreatic cancers, prostate cancers, colorectal cancers, andnon-small-cell lung cancers. The other cancers associated withoverexpression of HER-2-neu are potentially treatable with HERCEPTIN.

Accordingly, our methods of diagnosis may be combined with detection ofoverexpression of Her2 in an individual. Likewise, the methods oftreatment described here may include administration of Herceptin to anindividual, in addition to decreasing activity, amount or expression ofTAZ. We therefore provide a combination of TAZ nucleic acid or TAZpolypeptide, together with an anti-Her2 antibody. We also provide acombination of an anti-TAZ antibody together with an anti-Her2 antibody.In some embodiments, the anti-Her2 antibody comprises Herceptin.

Screening for Anti-TAZ Agents

Identifying TAZ Modulators, Agonists and Antagonists

Antagonists, in particular, small molecules may be used to specificallyinhibit TAZ for use as anti-TAZ agents.

We therefore disclose TAZ antagonists and small molecule TAZ inhibitors,as well as assays for screening for these. Antagonists of TAZ may bescreened by detecting modulation, such as down regulation, of binding orother TAZ activity. Antagonists of TAZ may also be screened by detectingmodulation of binding between TAZ and a TAZ binding protein, such asTEAD1, TEAD2, TEAD3 or TEAD4.

We therefore provide a compound capable of down-regulating theexpression, amount or activity TAZ polypeptide. Such a compound may beused in the methods and compositions described here for treating orpreventing cancer, particularly breast cancer.

TAZ may therefore be used to assess the binding of small moleculesubstrates and ligands in, for example, cells, cell-free preparations,chemical libraries, and natural product mixtures. These substrates andligands may be natural substrates and ligands or may be structural orfunctional mimetics. See Coligan et al., Current Protocols in Immunology1(2):Chapter 5 (1991). Furthermore, screens may be conducted to identifyfactors which influence the expression of TAZ, in particular in breastcells.

In general, the assays for agonists and antagonists rely on determiningthe effect of candidate molecules on one or more activities of TAZ. Anassay may involve assaying TAZ activity in the presence of a candidatemolecule, and optionally in the absence of the candidate molecule, or inthe presence of a molecule known to inhibit or activate a TAZ activity.Assays or modulators of activity of TAZ may be detected by detectingbinding of TAZ with another entity, such as a TAZ binding protein.Examples of TAZ binding proteins include TEAD1, TEAD2, TEAD3 and TEAD4.Accordingly, a screen for a modulator of TAZ activity such as a TAZantagonist may be conducted by providing TAZ and a TEAD polypeptide anddetecting the binding between them, in the presence and absence of acandidate molecule. Molecules of interest are those that interrupt,diminish, abolish, disrupt or in any way modulate the binding betweenTAZ and a TEAD polypeptide.

We have demonstrated that expression of TAZ is increased in breastcancer cells; accordingly, control of TAZ expression may be employed totreat breast cancer and other cancers. Therefore, it is desirous to findcompounds and drugs which stimulate the expression and/or activity ofTAZ, or which can inhibit the function of this protein. In general,agonists and antagonists are employed for therapeutic and prophylacticpurposes for any known cancer, in particular, breast cancer.

By “down-regulation” we include any negative effect on the behaviourbeing studied; this may be total or partial. Thus, where binding isbeing detected, candidate antagonists are capable of reducing,ameliorating, or abolishing the binding between two entities. Thedown-regulation of binding (or any other activity) achieved by thecandidate molecule may be at least 10%, such as at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 90% or more compared to binding (or which ever activity)in the absence of the candidate molecule. Thus, a candidate moleculesuitable for use as an antagonist is one which is capable of reducing by10% more the binding or other activity.

The term “compound” refers to a chemical compound (naturally occurringor synthesised), such as a biological macromolecule (e.g., nucleic acid,protein, non-peptide, or organic molecule), or an extract made frombiological materials such as bacteria, plants, fungi, or animal(particularly mammalian) cells or tissues, or even an inorganic elementor molecule. The compound may be an antibody.

Examples of potential antagonists of TAZ include antibodies, smallmolecules, nucleotides and their analogues, including purines and purineanalogues, oligonucleotides or proteins which are closely related to abinding partner of TAZ, e.g., a fragment of the binding partner, orsmall molecules which bind to the TAZ polypeptide but do not elicit aresponse, so that the activity of the polypeptide is prevented, etc.

Screening Kits

The materials necessary for such screening to be conducted may bepackaged into a screening kit.

Such a screening kit is useful for identifying agonists, antagonists,ligands, receptors, substrates, enzymes, etc. for TAZ polypeptides orcompounds which decrease or enhance the production of TAZ. The screeningkit may comprise: (a) a TAZ polypeptide; (b) a recombinant cellexpressing a TAZ polypeptide; (c) an antibody to TAZ polypeptide; or (d)a TAZ binding protein such as TEAD1, TEAD2, TEAD3 or TEAD4. Thescreening kit may comprise a library. The screening kit may comprise anyone or more of the components needed for screening, as described below.The screening kit may optionally comprise instructions for use.

Screening kits may also be provided which are capable of detecting TAZexpression at the nucleic acid level. Such kits may comprise a primerfor amplification of TAZ, or a pair of primers for amplification. Theprimer or primers may be chosen from any suitable sequence, for examplea portion of the TAZ sequence. Methods of identifying primer sequencesare well known in the art, and the skilled person will be able to designsuch primers with ease. The kits may comprise a nucleic acid probe forTAZ expression, as described in this document. The kits may alsooptionally comprise instructions for use.

Rational Design

Rational design of candidate compounds likely to be able to interactwith TAZ may be based upon structural studies of the molecular shapes ofa TAZ polypeptide.

For example, we have established that residues 52 and 53 of TAZ areinvolved in binding between TAZ and TEAD polypeptides. The Examples showthat mutants of TAZ at either of these positions (e.g., F52A, F53A)disrupt the binding between TAZ and TEAD polypeptides, disrupt thenuclear localisation of TAZ and disrupt the oncongenic potential of TAZ.

Accordingly, molecules comprising the sequence of TAZ surroundingpositions 52 and 53 may be used as modulators of TAZ activity, such asby modulating the binding between TAZ and TEAD polypeptides, e.g, bycompetitive binding. Such molecules may include a peptide comprisingposition 52 or position 53 or both of TAZ.

Furthermore, a peptide from a TAZ binding region of a TEAD (includingTEAD1, TEAD2, TEAD3 or TEAD4) may be designed and tested for modulationof TAZ-TEAD binding activity.

A further means for determining which sites interact with specific otherproteins is a physical structure determination, e.g., X-raycrystallography or two-dimensional NMR techniques.

These will provide guidance as to which amino acid residues formmolecular contact regions. For a detailed description of proteinstructural determination, see, e.g., Blundell and Johnson (1976) ProteinCrystallography, Academic Press, New York.

Polypeptide Binding Assays

Modulators and antagonists of TAZ activity or expression may beidentified by any means known in the art.

In their simplest form, the assays may simply comprise the steps ofmixing a candidate compound with a solution containing a TAZ polypeptideto form a mixture, measuring activity of TAZ polypeptide in the mixture,and comparing the activity of the mixture to a standard.

Furthermore, molecules may be identified by their binding to TAZ, in anassay which detects binding between TAZ and the putative molecule.

One type of assay for identifying substances that bind to a TAZpolypeptide described here involves contacting the TAZ polypeptide,which is immobilised on a solid support, with a non-immobilisedcandidate substance determining whether and/or to what extent the TAZpolypeptide of interest and candidate substance bind to each other.Alternatively, the candidate substance may be immobilised and the TAZpolypeptide as set out in this document non-immobilised.

The binding of the substance to the TAZ polypeptide can be transient,reversible or permanent. The substance may bind to the polypeptide witha Kd value which is lower than the Kd value for binding to controlpolypeptides (e.g., polypeptides known to not be involved in cancergrowth or progression). The Kd value of the substance may be 2 fold lessthan the Kd value for binding to control polypeptides, such as a Kdvalue 100 fold less or a Kd 1000 fold less than that for binding to thecontrol polypeptide.

In an example assay method, the TAZ polypeptide may be immobilised onbeads such as agarose beads. Typically this may be achieved byexpressing the TAZ polypeptide as a GST-fusion protein in bacteria,yeast or higher eukaryotic cell lines and purifying the GST-TAZ fusionprotein from crude cell extracts using glutathione-agarose beads (Smithand Johnson, 1988; Gene 67(10):31-40). As a control, binding of thecandidate substance, which is not a GST-fusion protein, to animmobilised polypeptide may be determined in the absence of the TAZpolypeptide. The binding of the candidate substance to the immobilisedTAZ polypeptide may then be determined. This type of assay is known inthe art as a GST pulldown assay. Again, the candidate substance may beimmobilised and the TAZ polypeptide non-immobilised.

It is also possible to perform this type of assay using differentaffinity purification systems for immobilising one of the components,for example Ni-NTA agarose and histidine-tagged components.

Binding of the polypeptide to the candidate substance may be determinedby a variety of methods well-known in the art. For example, thenon-immobilised component may be labeled (with for example, aradioactive label, an epitope tag or an enzyme-antibody conjugate).Alternatively, binding may be determined by immunological detectiontechniques. For example, the reaction mixture can be Western blotted andthe blot probed with an antibody that detects the non-immobilisedcomponent. ELISA techniques may also be used.

Candidate substances are typically added to a final concentration offrom 1 to 1000 nmol/ml, such as from 1 to 100 nmol/ml. In the case ofantibodies, the final concentration used is typically from 100 to 500μg/ml, such as from 200 to 300 μg/ml.

Modulators and antagonists of TAZ may also be identified by detectingmodulation of binding between TAZ and any molecule to which thispolypeptide binds, or modulation of any activity consequential on suchbinding or release.

Cell Based Assays

A cell based assay may simply test binding of a candidate compoundwherein adherence to the cells bearing the TAZ polypeptide is detectedby means of a label directly or indirectly associated with the candidatecompound or in an assay involving competition with a labeled competitor.

Further, these assays may test whether the candidate compound results ina signal generated by binding to the TAZ polypeptide, using detectionsystems appropriate to the cells bearing the polypeptides at theirsurfaces. Inhibitors of activation are generally assayed in the presenceof a known agonist and the effect on activation by the agonist by thepresence of the candidate compound is observed. Such a signal couldinclude nuclear localisation, which may be assayed as described in theExamples. Another signal which may be detected is oncogenic activity,which may be assayed by a soft agar assay, as described in the Examples.

Another method of screening compounds utilises eukaryotic or prokaryotichost cells which are stably transformed with recombinant DNA moleculesexpressing a library of compounds. Such cells, either in viable or fixedform, can be used for standard binding-partner assays. See also Parce etal. (1989) Science 246:243-247; and Owicki et al. (1990) Proc. Nat'lAcad. Sci. USA 87; 4007-4011, which describe sensitive methods to detectcellular responses.

Competitive assays are particularly useful, where the cells expressingthe library of compounds are contacted or incubated with a labelledantibody known to bind to a TAZ polypeptide, such as ¹²⁵I-antibody, anda test sample such as a candidate compound whose binding affinity to thebinding composition is being measured. The bound and free labelledbinding partners for the TAZ polypeptide are then separated to assessthe degree of binding. The amount of test sample bound is inverselyproportional to the amount of labelled antibody binding to the TAZpolypeptide.

Any one of numerous techniques can be used to separate bound from freebinding partners to assess the degree of binding. This separation stepcould typically involve a procedure such as adhesion to filters followedby washing, adhesion to plastic following by washing, or centrifugationof the cell membranes.

The assays may involve exposing a candidate molecule to a cell, such asa breast cell, and assaying expression of TAZ by any suitable means.Molecules which down-regulate the expression of TAZ in such assays maybe optionally chosen for further study, and used as drugs todown-regulate TAZ expression. Such drugs may be usefully employed totreat or prevent breast cancer.

cDNA encoding TAZ protein and antibodies to the proteins may also beused to configure assays for detecting the effect of added compounds onthe production of TAZ mRNA and protein in cells. For example, an ELISAmay be constructed for measuring secreted or cell associated levels ofTAZ polypeptide using monoclonal and polyclonal antibodies by standardmethods known in the art, and this can be used to discover agents whichmay inhibit or enhance the production of TAZ protein (also calledantagonist or agonist, respectively) from suitably manipulated cells ortissues. Standard methods for conducting screening assays are wellunderstood in the art.

Activity Assays

Assays to detect modulators or antagonists typically involve detectingmodulation of any activity of TAZ, in the presence, optionally togetherwith detection of modulation of activity in the absence, of a candidatemolecule.

The activity that may be detected can comprise any TAZ-dependentactivity, such as binding activity. TAZ is known to bind to SLC9A3R2 viathe PDZ motif at the plasma membrane, and binding activity of TAZ toSLC9A3R2 may be assayed by means known in the art, for example,GST-pulldown assays. One of TAZ and SLC9A3R2 may be immobilised and theother radiolabelled. Binding of TAZ to SLC9A3R2 may then be detected byassaying captured radioactivity on exposure of TAZ to SLC9A3R2.

Similarly, TAZ is known to bind to YWHAZ in vivo and in vitro throughthe phosphoserine-binding motif RSHSSP (SEQ ID NO: 7). Accordingly,detection of binding of TAZ to YWHAZ may be detected through means knownin the art, such as the techniques described above, and modulation ofsuch binding may be assayed to detect modulators or antagonists of TAZ.

TAZ is shown to bind to TEAD polypeptides, including TEAD1, TEAD2, TEAD3and TEAD4. Therefore, binding between TAZ and TEAD polypeptides may bedetected by means known in the art, such as the techniques described inthis document, and modulation of such binding may be assayed to detectmodulators or antagonists of TAZ.

Assays which detect specific biological activities of TAZ may also beused. The assays typically involve contacting a candidate molecule(e.g., in the form of a library) with TAZ whether in the form of apolypeptide, a nucleic acid encoding the polypeptide, or a cell,organelle, extract, or other material comprising such, with a candidatemodulator. The relevant activity of TAZ (as described below) may bedetected, to establish whether the presence of the candidate modulatorhas any effect.

Alternatively, or in addition, assaying of the binding between TAZ and14-3-3 as described by Kanai (2000) may be used to detect modulators ofTAZ. Hong et al. (2005) describes assays that detect TAZ-dependentco-activation of RUNX2-dependent gene transcription. Murakami et al.(2005) describes transactivation of TBX5 by TAZ.

The assays described by Kanai (2000), Hong et al (2005) or Murakami etal. (2005) may be performed in the presence or absence of a candidatemodulator and the appropriate activity detected to detect modulation ofTAZ activity and hence identification of a candidate modulator and/orantagonist of TAZ.

Promoter binding assays to detect candidate modulators which bind toand/or affect the transcription or expression of TAZ may also be used.Candidate modulators may then be chosen for further study, or isolatedfor use. Details of such screening procedures are well known in the art,and are for example described in, Handbook of Drug Screening, edited byRamakrishna Seethala, Prabhavathi B. Fernandes (2001, New York, N.Y.,Marcel Dekker, ISBN 0-8247-0562-9).

The screening methods described here may employ in vivo assays, althoughthey may be configured for in vitro use. In vivo assays generallyinvolve exposing a cell comprising TAZ to the candidate molecule. In invitro assays, TAZ is exposed to the candidate molecule, optionally inthe presence of other components, such as crude or semi-purified cellextract, or purified proteins. Where in vitro assays are conducted,these may employ arrays of candidate molecules (for example, an arrayedlibrary). In vivo assays may be employed. Therefore, the TAZ polypeptidemay be comprised in a cell, such as heterologously. Such a cell may be atransgenic cell, which has been engineered to express TAZ as describedabove.

Where an extract is employed, it may comprise a cytoplasmic extract or anuclear extract, methods of preparation of which are well known in theart.

It will be appreciated that any component of a cell comprising TAZ maybe employed, such as an organelle. One embodiment utilises a cytoplasmicor nuclear preparation, e.g., comprising a cell nucleus which comprisesTAZ as described. The nuclear preparation may comprise one or morenuclei, which may be permeabilised or semi-permeabilised, by detergenttreatment, for example.

Thus, in a specific embodiment, an assay format may include thefollowing: a multiwell microtitre plate is set up to include one or morecells expressing TAZ polypeptide in each well; individual candidatemolecules, or pools of candidate molecules, derived for example from alibrary, may be added to individual wells and modulation of TAZ activitymeasured. Where pools are used, these may be subdivided in to furtherpools and tested in the same manner. TAZ activity, for example bindingactivity or transcriptional co-activation activity, as describedelsewhere in this document may then be assayed.

Alternatively or in addition to the assay methods described above,“subtractive” procedures may also be used to identify modulators orantagonists of TAZ. Under such “subtractive” procedures, a plurality ofmolecules is provided, which comprises one or more candidate moleculescapable of functioning as a modulator (e.g., cell extract, nuclearextract, library of molecules, etc), and one or more components isremoved, depleted or subtracted from the plurality of molecules. The“subtracted” extract, etc, is then assayed for activity, by exposure toa cell comprising TAZ (or a component thereof) as described.

Thus, for example, an ‘immunodepletion’ assay may be conducted toidentify such modulators as follows. A cytoplasmic or nuclear extractmay be prepared from a cell. The extract may be depleted or fractionatedto remove putative modulators, such as by use of immunodepletion withappropriate antibodies. If the extract is depleted of a modulator, itwill lose the ability to affect TAZ function or activity or expression.A series of subtractions and/or depletions may be required to identifythe modulators or antagonists.

It will also be appreciated that the above “depletion” or “subtraction”assay may be used as a preliminary step to identify putative modulatoryfactors for further screening. Furthermore, or alternatively, the“depletion” or “subtraction” assay may be used to confirm the modulatoryactivity of a molecule identified by other means (for example, a“positive” screen as described elsewhere in this document) as a putativemodulator.

Candidate molecules subjected to the assay and which are found to be ofinterest may be isolated and further studied. Methods of isolation ofmolecules of interest will depend on the type of molecule employed,whether it is in the form of a library, how many candidate molecules arebeing tested at any one time, whether a batch procedure is beingfollowed, etc.

The candidate molecules may be provided in the form of a library. In oneembodiment, more than one candidate molecule may be screenedsimultaneously. A library of candidate molecules may be generated, forexample, a small molecule library, a polypeptide library, a nucleic acidlibrary, a library of compounds (such as a combinatorial library), alibrary of antisense molecules such as antisense DNA or antisense RNA,an antibody library etc, by means known in the art. Such libraries aresuitable for high-throughput screening. Different cells comprising TAZmay be exposed to individual members of the library, and effect on theTAZ activity determined. Array technology may be employed for thispurpose. The cells may be spatially separated, for example, in wells ofa microtitre plate.

In an embodiment, a small molecule library is employed. By a “smallmolecule”, we refer to a molecule whose molecular weight may be lessthan about 50 kDa. In particular embodiments, a small molecule may havea molecular weight which is less than about 30 kDa, such as less thanabout 15 kDa or less than 10 kDa or so. Libraries of such smallmolecules, here referred to as “small molecule libraries” may containpolypeptides, small peptides, for example, peptides of 20 amino acids orfewer, for example, 15, 10 or 5 amino acids, simple compounds, etc.

Alternatively or in addition, a combinatorial library, as described infurther detail below, may be screened for modulators or antagonists ofTAZ. Assays for TAZ activity are described above.

Libraries

Libraries of candidate molecules, such as libraries of polypeptides ornucleic acids, may be employed in the screens for TAZ antagonists andinhibitors described here. Such libraries are exposed to TAZ protein,and their effect, if any, on the activity of the protein determined.

Selection protocols for isolating desired members of large libraries areknown in the art, as typified by phage display techniques. Such systems,in which diverse peptide sequences are displayed on the surface offilamentous bacteriophage (Scott and Smith (1990 supra), have provenuseful for creating libraries of antibody fragments (and the nucleotidesequences that encoding them) for the in vitro selection andamplification of specific antibody fragments that bind a target antigen.The nucleotide sequences encoding the V_(H) and V_(L) regions are linkedto gene fragments which encode leader signals that direct them to theperiplasmic space of E. coli and as a result the resultant antibodyfragments are displayed on the surface of the bacteriophage, typicallyas fusions to bacteriophage coat proteins (e.g., pIII or pVIII).Alternatively, antibody fragments are displayed externally on lambdaphage capsids (phagebodies). An advantage of phage-based display systemsis that, because they are biological systems, selected library memberscan be amplified simply by growing the phage containing the selectedlibrary member in bacterial cells. Furthermore, since the nucleotidesequence that encodes the polypeptide library member is contained on aphage or phagemid vector, sequencing, expression and subsequent geneticmanipulation is relatively straightforward.

Methods for the construction of bacteriophage antibody display librariesand lambda phage expression libraries are well known in the art(McCafferty et al. (1990) supra; Kang et al. (1991) Proc. Natl. Acad.Sci. U.S.A., 88: 4363; Clackson et al. (1991) Nature, 352: 624; Lowmanet al. (1991) Biochemistry, 30: 10832; Burton et al. (1991) Proc. Natl.Acad. Sci U.S.A., 88: 10134; Hoogenboom et al. (1991) Nucleic AcidsRes., 19: 4133; Chang et al. (1991) J. Immunol., 147: 3610; Breitling etal. (1991) Gene, 104: 147; Marks et al. (1991) supra; Barbas et al.(1992) supra; Hawkins and Winter (1992) J. Immunol., 22: 867; Marks etal., 1992, J. Biol. Chem., 267: 16007; Lerner et al. (1992) Science,258: 1313, incorporated herein by reference). Such techniques may bemodified if necessary for the expression generally of polypeptidelibraries.

One particularly advantageous approach has been the use of scFvphage-libraries (Bird, R. E., et al. (1988) Science 242: 423-6, Hustonet al., 1988, Proc. Natl. Acad. Sci U.S.A., 85: 5879-5883; Chaudhary etal. (1990) Proc. Natl. Acad. Sci U.S.A., 87: 1066-1070; McCafferty etal. (1990) supra; Clackson et al. (1991) supra; Marks et al. (1991)supra; Chiswell et al. (1992) Trends Biotech., 10: 80; Marks et al.(1992) supra). Various embodiments of scFv libraries displayed onbacteriophage coat proteins have been described. Refinements of phagedisplay approaches are also known, for example as described inWO96/06213 and WO92/01047 (Medical Research Council et al.) andWO97/08320 (Morphosys, supra), which are incorporated herein byreference.

Alternative library selection technologies include bacteriophage lambdaexpression systems, which may be screened directly as bacteriophageplaques or as colonies of lysogens, both as previously described (Huseet al. (1989) Science, 246: 1275; Caton and Koprowski (1990) Proc. Natl.Acad. Sci. U.S.A., 87; Mullinax et al. (1990) Proc. Natl. Acad. Sci.U.S.A., 87: 8095; Persson et al. (1991) Proc. Natl. Acad. Sci. U.S.A.,88: 2432) and are of use in the methods and compositions described here.These expression systems may be used to screen a large number ofdifferent members of a library, in the order of about 10⁶ or even more.Other screening systems rely, for example, on direct chemical synthesisof library members. One early method involves the synthesis of peptideson a set of pins or rods, such as described in WO84/03564. A similarmethod involving peptide synthesis on beads, which forms a peptidelibrary in which each bead is an individual library member, is describedin U.S. Pat. No. 4,631,211 and a related method is described inWO92/00091. A significant improvement of the bead-based methods involvestagging each bead with a unique identifier tag, such as anoligonucleotide, so as to facilitate identification of the amino acidsequence of each library member. These improved bead-based methods aredescribed in WO93/06121.

Another chemical synthesis method involves the synthesis of arrays ofpeptides (or peptidomimetics) on a surface in a manner that places eachdistinct library member (e.g., unique peptide sequence) at a discrete,predefined location in the array. The identity of each library member isdetermined by its spatial location in the array. The locations in thearray where binding interactions between a predetermined molecule (e.g.,a receptor) and reactive library members occur is determined, therebyidentifying the sequences of the reactive library members on the basisof spatial location. These methods are described in U.S. Pat. No.5,143,854; WO90/15070 and WO92/10092; Fodor et al. (1991) Science, 251:767; Dower and Fodor (1991) Ann. Rep. Med. Chem., 26: 271.

Other systems for generating libraries of polypeptides or nucleotidesinvolve the use of cell-free enzymatic machinery for the in vitrosynthesis of the library members. In one method, RNA molecules areselected by alternate rounds of selection against a target ligand andPCR amplification (Tuerk and Gold (1990) Science, 249: 505; Ellingtonand Szostak (1990) Nature, 346: 818). A similar technique may be used toidentify DNA sequences which bind a predetermined human transcriptionfactor (Thiesen and Bach (1990) Nucleic Acids Res., 18: 3203; Beaudryand Joyce (1992) Science, 257: 635; WO92/05258 and WO92/14843). In asimilar way, in vitro translation can be used to synthesise polypeptidesas a method for generating large libraries. These methods whichgenerally comprise stabilised polysome complexes, are described furtherin WO88/08453, WO90/05785, WO90/07003, WO91/02076, WO91/05058, andWO92/02536. Alternative display systems which are not phage-based, suchas those disclosed in WO95/22625 and WO95/11922 (Affymax) use thepolysomes to display polypeptides for selection. These and all theforegoing documents also are incorporated herein by reference.

The library may in particular comprise a library of zinc fingers; zincfingers are known in the art and act as transcription factors. Suitablezinc finger libraries are disclosed in, for example, WO 96/06166 and WO98/53057. Construction of zinc finger libraries may utilise rules fordetermining interaction with specific DNA sequences, as disclosed in forexample WO 98/53058 and WO 98/53060. Zinc fingers capable of interactingspecifically with methylated DNA are disclosed in WO 99/47656. The abovezinc finger libraries may be immobilised in the form of an array, forexample as disclosed in WO 01/25417.

Combinatorial Libraries

Libraries, in particular, libraries of candidate molecules, may suitablybe in the form of combinatorial libraries (also known as combinatorialchemical libraries).

A “combinatorial library”, as the term is used in this document, is acollection of multiple species of chemical compounds that consist ofrandomly selected subunits. Combinatorial libraries may be screened formolecules which are capable of inhibiting TAZ.

Various combinatorial libraries of chemical compounds are currentlyavailable, including libraries active against proteolytic andnon-proteolytic enzymes, libraries of agonists and antagonists ofG-protein coupled receptors (GPCRs), libraries active against non-GPCRtargets (e.g., integrins, ion channels, domain interactions, nuclearreceptors, and transcription factors) and libraries of whole-celloncology and anti-infective targets, among others. A comprehensivereview of combinatorial libraries, in particular their construction anduses is provided in Dolle and Nelson (1999), Journal of CombinatorialChemistry, Vol 1 No 4, 235-282. Reference is also made to Combinatorialpeptide library protocols (edited by Shmuel Cabilly, Totowa, N.J.:Humana Press, c1998. Methods in Molecular Biology v. 87). Specificcombinatorial libraries and methods for their construction are disclosedin U.S. Pat. No. 6,168,914 (Campbell, et al), as well as in Baldwin etal. (1995), “Synthesis of a Small Molecule Library Encoded withMolecular Tags,” J. Am. Chem. Soc. 117:5588-5589, and in the referencesmentioned in those documents.

In one embodiment, the combinatorial library which is screened is onewhich is designed to potentially include molecules which interact with acomponent of the cell to influence gene expression. For example,combinatorial libraries against chromatin structural proteins may bescreened. Other libraries which are useful for this embodiment includecombinatorial libraries against histone modification enzymes (e.g.,histone acetylation or histone methylation enzymes), or DNAmodification, for example, DNA methylation or demethylation.

Further references describing chemical combinatorial libraries, theirproduction and use include those available from the URLhttp://www.netsci.org/Science/Combichem/, including The ChemicalGeneration of Molecular Diversity. Michael R. Pavia, SphinxPharmaceuticals, A Division of Eli Lilly (Published July, 1995);Combinatorial Chemistry: A Strategy for the Future—MDL InformationSystems discusses the role its Project Library plays in managingdiversity libraries (Published July, 1995); Solid Support CombinatorialChemistry in Lead Discovery and SAR Optimization, Adnan M. M. Mjalli andBarry E. Toyonaga, Ontogen Corporation (Published July, 1995);Non-Peptidic Bradykinin Receptor Antagonists From a StructurallyDirected Non-Peptide Library. Sarvajit Chakravarty, Babu J. Mavunkel,Robin Andy, Donald J. Kyle*, Scios Nova Inc. (Published July, 1995);Combinatorial Chemistry Library Design using Pharmacophore DiversityKeith Davies and Clive Briant, Chemical Design Ltd. (Published July,1995); A Database System for Combinatorial Synthesis Experiments—CraigJames and David Weininger, Daylight Chemical Information Systems, Inc.(Published July, 1995); An Information Management Architecture forCombinatorial Chemistry, Keith Davies and Catherine White, ChemicalDesign Ltd. (Published July, 1995); Novel Software Tools for AddressingChemical Diversity, R. S. Pearlman, Laboratory for Molecular Graphicsand Theoretical Modeling, College of Pharmacy, University of Texas(Published June/July, 1996); Opportunities for Computational ChemistsAfforded by the New Strategies in Drug Discovery: An Opinion, YvonneConnolly Martin, Computer Assisted Molecular Design Project, AbbottLaboratories (Published June/July, 1996); Combinatorial Chemistry andMolecular Diversity Course at the University of Louisville: ADescription, Arno F. Spatola, Department of Chemistry, University ofLouisville (Published June/July, 1996); Chemically Generated ScreeningLibraries: Present and Future. Michael R. Pavia, Sphinx Pharmaceuticals,A Division of Eli Lilly (Published June/July, 1996); Chemical StrategiesFor Introducing Carbohydrate Molecular Diversity Into The Drug DiscoveryProcess. Michael J. Sofia, Transcell Technologies Inc. (PublishedJune/July, 1996); Data Management for Combinatorial Chemistry. MaryjoZaborowski, Chiron Corporation and Sheila H. DeWitt, Parke-DavisPharmaceutical Research, Division of Warner-Lambert Company (PublishedNovember, 1995); and The Impact of High Throughput Organic Synthesis onR&D in Bio-Based Industries, John P. Devlin (Published March, 1996).

Techniques in combinatorial chemistry are gaining wide acceptance amongmodern methods for the generation of new pharmaceutical leads (Gallop,M. A. et al., 1994, J. Med. Chem. 37:1233-1251; Gordon, E. M. et al.,1994, J. Med. Chem. 37:1385-1401.). One combinatorial approach in use isbased on a strategy involving the synthesis of libraries containing adifferent structure on each particle of the solid phase support,interaction of the library with a soluble receptor, identification ofthe ‘bead’ which interacts with the macromolecular target, anddetermination of the structure carried by the identified ‘bead’ (Lam, K.S. et al., 1991, Nature 354:82-84). An alternative to this approach isthe sequential release of defined aliquots of the compounds from thesolid support, with subsequent determination of activity in solution,identification of the particle from which the active compound wasreleased, and elucidation of its structure by direct sequencing (Salmon,S. E. et al., 1993, Proc. Natl. Acad. Sci. USA 90:11708-11712), or byreading its code (Kerr, J. M. et al., 1993, J. Am. Chem. Soc.115:2529-2531; Nikolaiev, V. et al., 1993, Pept. Res. 6:161-170;Ohlmeyer, M. H. J. et al., 1993, Proc. Natl. Acad. Sci. USA90:10922-10926).

Soluble random combinatorial libraries may be synthesized using a simpleprinciple for the generation of equimolar mixtures of peptides which wasfirst described by Furka (Furka, A. et al., 1988, Xth InternationalSymposium on Medicinal Chemistry, Budapest 1988; Furka, A. et al., 1988,14th International Congress of Biochemistry, Prague 1988; Furka, A. etal., 1991, Int. J. Peptide Protein Res. 37:487-493). The construction ofsoluble libraries for iterative screening has also been described(Houghten, R. A. et al. 1991, Nature 354:84-86). K. S. Lam disclosed thenovel and unexpectedly powerful technique of using insoluble randomcombinatorial libraries. Lam synthesized random combinatorial librarieson solid phase supports, so that each support had a test compound ofuniform molecular structure, and screened the libraries without priorremoval of the test compounds from the support by solid phase bindingprotocols (Lam, K. S. et al., 1991, Nature 354:82-84).

Thus, a library of candidate molecules may be a synthetic combinatoriallibrary (e.g., a combinatorial chemical library), a cellular extract, abodily fluid (e.g., urine, blood, tears, sweat, or saliva), or othermixture of synthetic or natural products (e.g., a library of smallmolecules or a fermentation mixture).

A library of molecules may include, for example, amino acids,oligopeptides, polypeptides, proteins, or fragments of peptides orproteins; nucleic acids (e.g., antisense; DNA; RNA; or peptide nucleicacids, PNA); aptamers; or carbohydrates or polysaccharides. Each memberof the library can be singular or can be a part of a mixture (e.g., acompressed library). The library may contain purified compounds or canbe “dirty” (i.e., containing a significant quantity of impurities).

Commercially available libraries (e.g., from Affymetrix, ArQule, NeoseTechnologies, Sarco, Ciddco, Oxford Asymmetry, Maybridge, Aldrich,Panlabs, Pharmacopoeia, Sigma, or Tripose) may also be used with themethods described here.

In addition to libraries as described above, special libraries calleddiversity files can be used to assess the specificity, reliability, orreproducibility of the new methods. Diversity files contain a largenumber of compounds (e.g., 1000 or more small molecules) representativeof many classes of compounds that could potentially result innonspecific detection in an assay. Diversity files are commerciallyavailable or can also be assembled from individual compoundscommercially available from the vendors listed above.

Anti-TAZ Antibodies

Anti-TAZ agents, including antagonists or modulators of TAZ, which maybe used to regulate the activity of this protein (for example, formethods of treating or preventing diseases such as cancer as describedin this document) may include antibodies against the TAZ protein.

We therefore provide for antibodies which bind to a TAZ polypeptide,fragment, homologue, variant or derivative thereof. Such antibodies areuseful in detecting TAZ expression, and in particular in diagnosing aTAZ associated disease such as breast cancer. Other antibodies includethose which have therapeutic activity, i.e., which are may be used in atherapeutic manner to treat, manage or prevent any TAZ associateddisease, including breast cancer.

Examples of antibodies capable of binding to TAZ include rabbit anti-TAZantibody against amino acids 160-229 of TAZ, rabbit anti-TAZ antibody(catalogue number 2149S, Cell Signaling Technology, Danvers, Mass.,USA), rabbit polyclonal anti-TAZ antibody (catalogue numberNB110-58359SS, Novus Biological, Littleton, Colo., USA), MouseMonoclonal anti-TAZ [1B10] (catalogue number H00006901-M12, NovusBiological, Littleton, Colo., USA), Rabbit anti-Human TAZ PolyclonalAntibody (catalogue number LS-B94, LifeSpan Biosciences, Inc., Seattle,Wash., USA) or p-TAZ (Ser 89)-R (catalogue number sc-17610-R, Santa CruzBiotechnology, Santa Cruz, Calif., USA).

Furthermore, antibodies which are specific for TAZ may be generatedagainst any suitable epitope, for example, an epitope derived from theTAZ protein. The sequence of a suitable fragment of TAZ may compriseresidues 160-229 of TAZ and any epitope from this sequence may be usedfor the generation of specific TAZ antibodies.

For the purposes of this document, the term “antibody” refers tocomplete antibodies or antibody fragments capable of binding to aselected target. Unless specified to the contrary, the term includes butis not limited to, polyclonal, monoclonal, natural or engineeredantibodies including chimeric, CDR-grafted and humanised antibodies, andartificially selected antibodies produced using phage display oralternative techniques. The term also includes single chain, Fabfragments and fragments produced by a Fab expression library. Suchfragments include fragments of whole antibodies which retain theirbinding activity for a target substance, Fv, F(ab′) and F(ab′)₂fragments, as well as single chain antibodies (scFv), fusion proteinsand other synthetic proteins which comprise the antigen-binding site ofthe antibody. Small fragments, such as Fv and ScFv, possess advantageousproperties for diagnostic and therapeutic applications on account oftheir small size and consequent superior tissue distribution.

The antibodies and fragments thereof may be humanised antibodies, forexample as described in EP-A-239400. Furthermore, antibodies with fullyhuman variable regions (or their fragments), for example, as describedin U.S. Pat. Nos. 5,545,807 and 6,075,181 may also be used. Neutralizingantibodies, i.e., those which inhibit any biological activity of TAZ,may be used for diagnostics and therapeutics.

The antibodies described here may be altered antibodies comprising aneffector protein such as a label. Labels which allow the imaging of thedistribution of the antibody in vivo or in vitro may be used. Suchlabels may be radioactive labels or radioopaque labels, such as metalparticles, which are readily visualisable within an embryo or a cellmass. Moreover, they may be fluorescent labels or other labels which arevisualisable on tissue samples.

Antibodies may be produced by standard techniques, such as byimmunisation or by using a phage display library. Such an antibody maybe capable of binding specifically to the TAZ protein or homologue,fragment, etc.

Polyclonal Antibodies

If polyclonal antibodies are desired, a selected mammal (e.g., mouse,rabbit, goat, horse, etc.) may be immunised with an immunogeniccomposition comprising a TAZ polypeptide or peptide. Depending on thehost species, various adjuvants may be used to increase immunologicalresponse. Such adjuvants include, but are not limited to, Freund's,mineral gels such as aluminum hydroxide, and surface active substancessuch as lysolecithin, pluronic polyols, polyanions, peptides, oilemulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG (BacilliCalmette-Guerin) and Corynebacterium parvum are potentially useful humanadjuvants which may be employed if purified the substance amino acidsequence is administered to immunologically compromised individuals forthe purpose of stimulating systemic defence.

Serum from the immunised animal is collected and treated according toknown procedures. If serum containing polyclonal antibodies to anepitope obtainable from a TAZ polypeptide contains antibodies to otherantigens, the polyclonal antibodies can be purified by immunoaffinitychromatography. Techniques for producing and processing polyclonalantisera are known in the art. In order that such antibodies may bemade, we also provide TAZ amino acid sequences or fragments thereofhaptenised to another amino acid sequence for use as immunogens inanimals or humans.

Monoclonal Antibodies

Monoclonal antibodies directed against epitopes obtainable from a TAZpolypeptide or peptide can also be readily produced by one skilled inthe art. The general methodology for making monoclonal antibodies byhybridomas is well known. Immortal antibody-producing cell lines can becreated by cell fusion, and also by other techniques such as directtransformation of B lymphocytes with oncogenic DNA, or transfection withEpstein-Barr virus. Panels of monoclonal antibodies produced againstorbit epitopes can be screened for various properties; i.e., for isotypeand epitope affinity.

Monoclonal antibodies may be prepared using any technique which providesfor the production of antibody molecules by continuous cell lines inculture. These include, but are not limited to, the hybridoma techniqueoriginally described by Koehler and Milstein (1975 Nature 256:495-497),the trioma technique, the human B-cell hybridoma technique (Kosbor et al(1983) Immunol Today 4:72; Cote et al (1983) Proc Natl Acad Sci80:2026-2030) and the EBV-hybridoma technique (Cole et al., MonoclonalAntibodies and Cancer Therapy, pp. 77-96, Alan R. Liss, Inc., 1985).

Recombinant DNA technology may be used to improve the antibodies asdescribed here. Thus, chimeric antibodies may be constructed in order todecrease the immunogenicity thereof in diagnostic or therapeuticapplications. Such techniques comprise splicing of mouse antibody genesto human antibody genes to obtain a molecule with appropriate antigenspecificity and biological activity (Morrison et al (1984) Proc NatlAcad Sci 81:6851-6855; Neuberger et al (1984) Nature 312:604-608; Takedaet al (1985) Nature 314:452-454). Moreover, immunogenicity may beminimised by humanising the antibodies by CDR grafting [see EuropeanPatent Application 0 239 400 (Winter)] and, optionally, frameworkmodification [EP 0 239 400].

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,779) can be adapted to produce thesubstance specific single chain antibodies.

Antibodies, both monoclonal and polyclonal, which are directed againstepitopes obtainable from a TAZ polypeptide or peptide are particularlyuseful in diagnosis. Monoclonal antibodies, in particular, may be usedto raise anti-idiotype antibodies. Anti-idiotype antibodies areimmunoglobulins which carry an “internal image” of the substance and/oragent against which protection is desired. Techniques for raisinganti-idiotype antibodies are known in the art. These anti-idiotypeantibodies may also be useful in therapy.

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inOrlandi et al (1989, Proc Natl Acad Sci 86: 3833-3837), and Winter G andMilstein C (1991; Nature 349:293-299).

Antibody fragments which contain specific binding sites for thepolypeptide or peptide may also be generated. For example, suchfragments include, but are not limited to, the F(ab′)₂ fragments whichcan be produced by pepsin digestion of the antibody molecule and the Fabfragments which can be generated by reducing the disulfide bridges ofthe F(ab′)₂ fragments. Alternatively, Fab expression libraries may beconstructed to allow rapid and easy identification of monoclonal Fabfragments with the desired specificity (Huse W D et al (1989) Science256:1275-128 1).

Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can also be adapted to produce single chain antibodies to TAZpolypeptides. Also, transgenic mice, or other organisms including othermammals, may be used to express humanized antibodies.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptide or to purify the polypeptides byaffinity chromatography.

Recombinant Techniques of Antibody Production

Recombinant DNA technology may be used to produce the antibodiesaccording to established procedure, in bacterial or mammalian cellculture. The selected cell culture system may secrete the antibodyproduct.

Therefore, we disclose a process for the production of an antibodycomprising culturing a host, e.g. E. coli or a mammalian cell, which hasbeen transformed with a hybrid vector comprising an expression cassettecomprising a promoter operably linked to a first DNA sequence encoding asignal peptide linked in the proper reading frame to a second DNAsequence encoding said antibody protein, and isolating said protein.

Multiplication of hybridoma cells or mammalian host cells in vitro iscarried out in suitable culture media, which are the customary standardculture media, for example Dulbecco's Modified Eagle Medium (DMEM) orRPMI 1640 medium, optionally replenished by a mammalian serum, e.g.foetal calf serum, or trace elements and growth sustaining supplements,e.g. feeder cells such as normal mouse peritoneal exudate cells, spleencells, bone marrow macrophages, 2-aminoethanol, insulin, transferrin,low density lipoprotein, oleic acid, or the like. Multiplication of hostcells which are bacterial cells or yeast cells is likewise carried outin suitable culture media known in the art, for example for bacteria inmedium LB, NZCYM, NZYM, NZM, Terrific Broth, SOB, SOC, 2×YT, or M9Minimal Medium, and for yeast in medium YPD, YEPD, Minimal Medium, orComplete Minimal Dropout Medium.

In vitro production provides relatively pure antibody preparations andallows scale-up to give large amounts of the desired antibodies.Techniques for bacterial cell, yeast or mammalian cell cultivation areknown in the art and include homogeneous suspension culture, e.g. in anairlift reactor or in a continuous stirrer reactor, or immobilised orentrapped cell culture, e.g. in hollow fibres, microcapsules, on agarosemicrobeads or ceramic cartridges.

Large quantities of the desired antibodies can also be obtained bymultiplying mammalian cells in vivo. For this purpose, hybridoma cellsproducing the desired antibodies are injected into histocompatiblemammals to cause growth of antibody-producing tumours. Optionally, theanimals are primed with a hydrocarbon, especially mineral oils such aspristane (tetramethyl-pentadecane), prior to the injection. After one tothree weeks, the antibodies are isolated from the body fluids of thosemammals. For example, hybridoma cells obtained by fusion of suitablemyeloma cells with antibody-producing spleen cells from Balb/c mice, ortransfected cells derived from hybridoma cell line Sp2/0 that producethe desired antibodies are injected intraperitoneally into Balb/c miceoptionally pre-treated with pristane, and, after one to two weeks,ascitic fluid is taken from the animals.

The foregoing, and other, techniques are discussed in, for example,Kohler and Milstein, (1975) Nature 256:495-497; U.S. Pat. No. 4,376,110;Harlow and Lane, Antibodies: a Laboratory Manual, (1988) Cold SpringHarbor, incorporated herein by reference. Techniques for the preparationof recombinant antibody molecules are described in the above referencesand also in, for example, EP 0623679; EP 0368684 and EP 0436597, whichare incorporated herein by reference.

The cell culture supernatants are screened for the desired antibodies,preferentially by immunofluorescent staining of PGCs or otherpluripotent cells, such as ES or EG cells, by immunoblotting, by anenzyme immunoassay, e.g. a sandwich assay or a dot-assay, or aradioimmunoassay.

For isolation of the antibodies, the immunoglobulins in the culturesupernatants or in the ascitic fluid may be concentrated, e.g. byprecipitation with ammonium sulphate, dialysis against hygroscopicmaterial such as polyethylene glycol, filtration through selectivemembranes, or the like. If necessary and/or desired, the antibodies arepurified by the customary chromatography methods, for example gelfiltration, ion-exchange chromatography, chromatography overDEAE-cellulose and/or (immuno-) affinity chromatography, e.g. affinitychromatography with the antigen, or fragments thereof, or withProtein-A.

Hybridoma cells secreting the monoclonal antibodies are also provided.Hybridoma cells may be genetically stable, secrete monoclonal antibodiesof the desired specificity and can be activated from deep-frozencultures by thawing and recloning.

Also included is a process for the preparation of a hybridoma cell linesecreting monoclonal antibodies directed to the TAZ polypeptide,characterised in that a suitable mammal, for example a Balb/c mouse, isimmunised with a one or more TAZ polypeptides, or antigenic fragmentsthereof; antibody-producing cells of the immunised mammal are fused withcells of a suitable myeloma cell line, the hybrid cells obtained in thefusion are cloned, and cell clones secreting the desired antibodies areselected. For example spleen cells of Balb/c mice immunised with TAZ arefused with cells of the myeloma cell line PAI or the myeloma cell lineSp2/0-Ag14, the obtained hybrid cells are screened for secretion of thedesired antibodies, and positive hybridoma cells are cloned.

We describe a process for the preparation of a hybridoma cell line,characterised in that Balb/c mice are immunised by injectingsubcutaneously and/or intraperitoneally between 10 and 10⁷ and 10⁸ cellsexpressing TAZ and a suitable adjuvant several times, e.g. four to sixtimes, over several months, e.g. between two and four months, and spleencells from the immunised mice are taken two to four days after the lastinjection and fused with cells of the myeloma cell line PAI in thepresence of a fusion promoter, such as polyethylene glycol. The myelomacells may be fused with a three- to twentyfold excess of spleen cellsfrom the immunised mice in a solution containing about 30% to about 50%polyethylene glycol of a molecular weight around 4000. After the fusionthe cells are expanded in suitable culture media as describedhereinbefore, supplemented with a selection medium, for example HATmedium, at regular intervals in order to prevent normal myeloma cellsfrom overgrowing the desired hybridoma cells.

Recombinant DNAs comprising an insert coding for a heavy chain variabledomain and/or for a light chain variable domain of antibodies directedto TAZ as described hereinbefore are also disclosed. By definition suchDNAs comprise coding single stranded DNAs, double stranded DNAsconsisting of said coding DNAs and of complementary DNAs thereto, orthese complementary (single stranded) DNAs themselves.

Furthermore, DNA encoding a heavy chain variable domain and/or for alight chain variable domain of antibodies directed to TAZ can beenzymatically or chemically synthesised DNA having the authentic DNAsequence coding for a heavy chain variable domain and/or for the lightchain variable domain, or a mutant thereof. A mutant of the authenticDNA is a DNA encoding a heavy chain variable domain and/or a light chainvariable domain of the above-mentioned antibodies in which one or moreamino acids are deleted or exchanged with one or more other amino acids.The modification(s) may be outside the CDRs of the heavy chain variabledomain and/or of the light chain variable domain of the antibody. Such amutant DNA is also intended to be a silent mutant wherein one or morenucleotides are replaced by other nucleotides with the new codons codingfor the same amino acid(s). Such a mutant sequence is also a degeneratedsequence. Degenerated sequences are degenerated within the meaning ofthe genetic code in that an unlimited number of nucleotides are replacedby other nucleotides without resulting in a change of the amino acidsequence originally encoded. Such degenerated sequences may be usefuldue to their different restriction sites and/or frequency of particularcodons which are preferred by the specific host, particularly E. coli,to obtain an optimal expression of the heavy chain murine variabledomain and/or a light chain murine variable domain.

The term mutant is intended to include a DNA mutant obtained by in vitromutagenesis of the authentic DNA according to methods known in the art.

For the assembly of complete tetrameric immunoglobulin molecules and theexpression of chimeric antibodies, the recombinant DNA inserts codingfor heavy and light chain variable domains are fused with thecorresponding DNAs coding for heavy and light chain constant domains,then transferred into appropriate host cells, for example afterincorporation into hybrid vectors.

Also disclosed are recombinant DNAs comprising an insert coding for aheavy chain murine variable domain of an antibody directed to TAZ fusedto a human constant domain g, for example γ1, γ2, γ3 or γ4, such as γ1or γ4. Likewise recombinant DNAs comprising an insert coding for a lightchain murine variable domain of an antibody directed to TAZ fused to ahuman constant domain κ or λ, such as κ are also disclosed.

In another embodiment, we disclose recombinant DNAs coding for arecombinant polypeptide wherein the heavy chain variable domain and thelight chain variable domain are linked by way of a spacer group,optionally comprising a signal sequence facilitating the processing ofthe antibody in the host cell and/or a DNA coding for a peptidefacilitating the purification of the antibody and/or a cleavage siteand/or a peptide spacer and/or an effector molecule.

The DNA coding for an effector molecule is intended to be a DNA codingfor the effector molecules useful in diagnostic or therapeuticapplications. Thus, effector molecules which are toxins or enzymes,especially enzymes capable of catalysing the activation of prodrugs, areparticularly indicated. The DNA encoding such an effector molecule hasthe sequence of a naturally occurring enzyme or toxin encoding DNA, or amutant thereof, and can be prepared by methods well known in the art.

Use

Anti-TAZ antibodies may be used in method of detecting a TAZ polypeptidepresent in biological samples by a method which comprises: (a) providingan anti-TAZ antibody; (b) incubating a biological sample with saidantibody under conditions which allow for the formation of anantibody-antigen complex; and (c) determining whether antibody-antigencomplex comprising said antibody is formed.

Suitable samples include extracts tissues such as brain, breast, ovary,lung, colon, pancreas, testes, liver, muscle and bone tissues or fromneoplastic growths derived from such tissues. In particular, a samplemay comprise a breast tissue, such as a breast tissue from an individualsuspected to be suffering from breast cancer.

Antibodies may be bound to a solid support and/or packaged into kits ina suitable container along with suitable reagents, controls,instructions and the like.

Antibody Delivery

The antibodies against the TAZ protein may be delivered into a cell bymeans of techniques known in the art, for example by the use ofliposomes, polymers, (e.g., polyethylene glycol (PEG),N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers, polyamidoamine(PAMAM) dendrimers, HEMA, linear polyamidoamine polymers etc) etc. Theimmunoglobulins and/or antibodies may also be delivered into cells asprotein fusions or conjugates with a protein capable of crossing theplasma membrane and/or the nuclear membrane. For example, theimmunoglobulin and/or target may be fused or conjugated to a domain orsequence from such a protein responsible for the translocationalactivity. Translocation domains and sequences may include domains andsequences from the HIV-1-trans-activating protein (Tat), DrosophilaAntennapedia homeodomain protein and the herpes simplex-1 virus VP22protein.

Pharmaceutical Compositions and Administration

While it is possible for the anti-TAZ agent, including an TAZ nucleicacid, polypeptide, fragment, homologue, variant or derivative thereof,modulator, agonist or antagonist, a structurally related compound, or anacidic salt of either to be administered alone, the active ingredientmay be formulated as a pharmaceutical formulation.

We therefore also disclose pharmaceutical compositions comprising ananti-TAZ agent. Such pharmaceutical compositions are useful for deliveryof the anti-TAZ agent such as in the form of a composition as described,to an individual for the treatment or alleviation of symptoms asdescribed.

A pharmaceutical composition in the context of the present document is acomposition of matter comprising at least an anti-TAZ agent as an activeingredient.

The pharmaceutical formulations comprise an effective amount of theanti-TAZ agent together with one or more pharmaceutically-acceptablecarriers. An “effective amount” is the amount sufficient to alleviate atleast one symptom of a disease as described.

The effective amount will vary depending upon the particular disease orsyndrome to be treated or alleviated, as well as other factors includingthe age and weight of the patient, how advanced the disease etc stateis, the general health of the patient, the severity of the symptoms, andwhether the anti-TAZ agent is being administered alone or in combinationwith other therapies.

Suitable pharmaceutically acceptable carriers are well known in the artand vary with the desired form and mode of administration of thepharmaceutical formulation. For example, they can include diluents orexcipients such as fillers, binders, wetting agents, disintegrators,surface-active agents, lubricants and the like. Typically, the carrieris a solid, a liquid or a vaporizable carrier, or a combination thereof.Each carrier should be “acceptable” in the sense of being compatiblewith the other ingredients in the formulation and not injurious to thepatient. The carrier should be biologically acceptable without elicitingan adverse reaction (e.g. immune response) when administered to thehost.

The active ingredient(s) of a pharmaceutical composition is contemplatedto exhibit therapeutic activity, for example, in the alleviation ofcancer, tumours, neoplasms and other related diseases. Dosage regimesmay be adjusted to provide the optimum therapeutic response. Forexample, several divided doses may be administered daily or the dose maybe proportionally reduced as indicated by the exigencies of thetherapeutic situation.

The active compound may be administered in a convenient manner such asby the oral, intravenous (where water soluble), intramuscular,subcutaneous, intranasal, intradermal or suppository routes orimplanting (e.g. using slow release molecules). Depending on the routeof administration, the active ingredient may be required to be coated ina material to protect said ingredients from the action of enzymes, acidsand other natural conditions which may inactivate said ingredient.

The anti-TAZ agent may be administered alone, or in combination withother therapeutic agents. Other therapeutic agents suitable for useherein are any compatible drugs that are effective for the intendedpurpose, or drugs that are complementary to the agent formulation. Theformulation utilized in a combination therapy may be administeredsimultaneously, or sequentially with other treatment, such that acombined effect is achieved.

Oral Administration

In some embodiments, the inhibitor of TAZ activity, expression or amountis provided as an oral composition and administered accordingly. Thedosage of the inhibitor of TAZ activity, expression or amount may bebetween about 1 mg/day to about 10 mg/day.

The pharmaceutical composition can be administered in an oralformulation in the form of tablets, capsules or solutions. An effectiveamount of the oral formulation is administered to patients 1 to 3 timesdaily until the symptoms of the disease alleviated.

The effective amount of agent depends on the age, weight and conditionof a patient. In general, the daily oral dose of agent is less than 1200mg, and more than 100 mg. The daily oral dose may be about 300-600 mg.Oral formulations are conveniently presented in a unit dosage form andmay be prepared by any method known in the art of pharmacy. Thecomposition may be formulated together with a suitable pharmaceuticallyacceptable carrier into any desired dosage form. Typical unit dosageforms include tablets, pills, powders, solutions, suspensions,emulsions, granules, capsules, suppositories. In general, theformulations are prepared by uniformly and intimately bringing intoassociation the agent composition with liquid carriers or finely dividedsolid carriers or both, and as necessary, shaping the product. Theactive ingredient can be incorporated into a variety of basic materialsin the form of a liquid, powder, tablets or capsules to give aneffective amount of active ingredient to treat the disease.

The composition may be suitably orally administered, for example, withan inert diluent or with an assimilable edible carrier, or it may beenclosed in hard or soft shell gelatin capsules, or it may be compressedinto tablets, or it may be incorporated directly with the food of thediet. For oral therapeutic administration, the active compound may beincorporated with excipients and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,and the like. The amount of active compound in such therapeuticallyuseful compositions in such that a suitable dosage will be obtained.

The tablets, troches, pills, capsules and the like may also contain thefollowing: a binder such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, lactose or saccharin may be added or a flavouring agent such aspeppermint, oil of wintergreen, or cherry flavouring. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier.

Various other materials may be present as coatings or to otherwisemodify the physical form of the dosage unit. For instance, tablets,pills, or capsules may be coated with shellac, sugar or both. A syrup orelixir may contain the active compound, sucrose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavouring such ascherry or orange flavour. Of course, any material used in preparing anydosage unit form should be pharmaceutically pure and substantiallynon-toxic in the amounts employed. In addition, the active compound maybe incorporated into sustained-release preparations and formulations.

Injectable or Intravenous Administration

In some embodiments, the anti-TAZ agent is provided as an injectable orintravenenous composition and administered accordingly. The dosage ofthe anti-TAZ agent inhibitor may be between about 5 mg/kg/2 weeks toabout 10 mg/kg/2 weeks. The anti-TAZ agent inhibitor may be provided ina dosage of between 10-300 mg/day, such as at least 30 mg/day, less than200 mg/day or between 30 mg/day to 200 mg/day.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. In all cases the form must be sterile and mustbe fluid to the extent that easy syringability exists. It must be stableunder the conditions of manufacture and storage and must be preservedagainst the contaminating action of microorganisms such as bacteria andfungi. The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyetheylene glycol, and the like), suitablemixtures thereof, and vegetable oils. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of superfactants.

Topical Administration

The pharmaceutical compositions disclosed here include those suitablefor topical and oral administration, with topical formulations beingpreferred where the tissue affected is primarily the skin or epidermis(for example, psoriasis, eczema and other epidermal diseases).

The topical formulations include those pharmaceutical forms in which thecomposition is applied externally by direct contact with the skinsurface to be treated. A conventional pharmaceutical form for topicalapplication includes a soak, an ointment, a cream, a lotion, a paste, agel, a stick, a spray, an aerosol, a bath oil, a solution and the like.Topical therapy is delivered by various vehicles, the choice of vehiclecan be important and generally is related to whether an acute or chronicdisease is to be treated. As an example, an acute skin proliferationdisease generally is treated with aqueous drying preparations, whereaschronic skin proliferation disease is treated with hydratingpreparations. Soaks are the easiest method of drying acute moisteruptions. Lotions (powder in water suspension) and solutions(medications dissolved in a solvent) are ideal for hairy andintertriginous areas. Ointments or water-in-oil emulsions, are the mosteffective hydrating agents, appropriate for dry scaly eruptions, but aregreasy and depending upon the site of the lesion sometimes undesirable.As appropriate, they can be applied in combination with a bandage,particularly when it is desirable to increase penetration of the agentcomposition into a lesion. Creams or oil-in-water emulsions and gels areabsorbable and are the most cosmetically acceptable to the patient.(Guzzo et al, in Goodman & Gilman's Pharmacological Basis ofTherapeutics, 9th Ed., p. 1593-15950 (1996)). Cream formulationsgenerally include components such as petroleum, lanolin, polyethyleneglycols, mineral oil, glycerin, isopropyl palmitate, glyceryl stearate,cetearyl alcohol, tocopheryl acetate, isopropyl myristate, lanolinalcohol, simethicone, carbomen, methylchlorisothiazolinone,methylisothiazolinone, cyclomethicone and hydroxypropyl methylcellulose,as well as mixtures thereof.

Other formulations for topical application include shampoos, soaps,shake lotions, and the like, particularly those formulated to leave aresidue on the underlying skin, such as the scalp (Arndt et al, inDermatology In General Medicine 2:2838 (1993)).

In general, the concentration of the composition in the topicalformulation is in an amount of about 0.5 to 50% by weight of thecomposition, such as about 1 to 30%, about 2-20%, or about 5-10%. Theconcentration used can be in the upper portion of the range initially,as treatment continues, the concentration can be lowered or theapplication of the formulation may be less frequent. Topicalapplications are often applied twice daily. However, once-dailyapplication of a larger dose or more frequent applications of a smallerdose may be effective. The stratum corneum may act as a reservoir andallow gradual penetration of a drug into the viable skin layers over aprolonged period of time.

In a topical application, a sufficient amount of active ingredient mustpenetrate a patient's skin in order to obtain a desired pharmacologicaleffect. It is generally understood that the absorption of drug into theskin is a function of the nature of the drug, the behaviour of thevehicle, and the skin. Three major variables account for differences inthe rate of absorption or flux of different topical drugs or the samedrug in different vehicles; the concentration of drug in the vehicle,the partition coefficient of drug between the stratum corneum and thevehicle and the diffusion coefficient of drug in the stratum corneum. Tobe effective for treatment, a drug must cross the stratum corneum whichis responsible for the barrier function of the skin. In general, atopical formulation which exerts a high in vitro skin penetration iseffective in vivo. Ostrenga et al (J. Pharm. Sci., 60:1175-1179 (1971)demonstrated that in vivo efficacy of topically applied steroids wasproportional to the steroid penetration rate into dermatomed human skinin vitro.

A skin penetration enhancer which is dermatologically acceptable andcompatible with the agent can be incorporated into the formulation toincrease the penetration of the active compound(s) from the skin surfaceinto epidermal keratinocytes. A skin enhancer which increases theabsorption of the active compound(s) into the skin reduces the amount ofagent needed for an effective treatment and provides for a longerlasting effect of the formulation. Skin penetration enhancers are wellknown in the art. For example, dimethyl sulfoxide (U.S. Pat. No.3,711,602); oleic acid, 1,2-butanediol surfactant (Cooper, J. Pharm.Sci., 73:1153-1156 (1984)); a combination of ethanol and oleic acid oroleyl alcohol (EP 267,617), 2-ethyl-1,3-hexanediol (WO 87/03490); decylmethyl sulphoxide and Azone® (Hadgraft, Eur. J. Drug. Metab.Pharmacokinet, 21:165-173 (1996)); alcohols, sulphoxides, fatty acids,esters, Azone®, pyrrolidones, urea and polyoles (Kalbitz et al,Pharmazie, 51:619-637 (1996));

Terpenes such as 1,8-cineole, menthone, limonene and nerolidol (Yamane,J. Pharmacy & Pharmocology, 47:978-989 (1995)); Azone® and Transcutol(Harrison et al, Pharmaceutical Res. 13:542-546 (1996)); and oleic acid,polyethylene glycol and propylene glycol (Singh et al, Pharmazie,51:741-744 (1996)) are known to improve skin penetration of an activeingredient.

Levels of penetration of an agent or composition can be determined bytechniques known to those of skill in the art. For example,radiolabeling of the active compound, followed by measurement of theamount of radiolabeled compound absorbed by the skin enables one ofskill in the art to determine levels of the composition absorbed usingany of several methods of determining skin penetration of the testcompound. Publications relating to skin penetration studies includeReinfenrath, W G and G S Hawkins. The Weaning Yorkshire Pig as an AnimalModel for Measuring Percutaneous Penetration. In: Swine in BiomedicalResearch (M. E. Tumbleson, Ed.) Plenum, New York, 1986, and Hawkins, G.S. Methodology for the Execution of In Vitro Skin PenetrationDeterminations. In: Methods for Skin Absorption, B W Kemppainen and W GReifenrath, Eds., CRC Press, Boca Raton, 1990, pp. 67-80; and W. G.Reifenrath, Cosmetics & Toiletries, 110:3-9 (1995).

For some applications, a long acting form of agent or composition may beadministered using formulations known in the arts, such as polymers. Theagent can be incorporated into a dermal patch (Junginger, H. E., in ActaPharmaceutica Nordica 4:117 (1992); Thacharodi et al, in Biomaterials16:145-148 (1995); Niedner R., in Hautarzt 39:761-766 (1988)) or abandage according to methods known in the arts, to increase theefficiency of delivery of the drug to the areas to be treated.

Optionally, the topical formulations described here can have additionalexcipients for example; preservatives such as methylparaben, benzylalcohol, sorbic acid or quaternary ammonium compound; stabilizers suchas EDTA, antioxidants such as butylated hydroxytoluene or butylatedhydroxanisole, and buffers such as citrate and phosphate.

Parenteral Administration

The active compound may also be administered parenterally orintraperitoneally. Dispersions can also be prepared in glycerol, liquidpolyethylene glycols, and mixtures thereof and in oils. In someembodiments, the dispersions may be prepared in 30% Capsitol (CyDex,Inc., Lenexa, Kans., USA). Capsitol is a polyanionic β-cyclodextrinderivative with a sodium sulfonate salt separated from the lipophiliccavity by a butyl ether spacer group, or sulfobutylether (SBE). Thecyclodextrin may be SBE7-β-CD.

Adjuvants

The composition may be administered in an adjuvant, co-administered withenzyme inhibitors or in liposomes. Adjuvant is used in its broadestsense and includes any immune stimulating compound such as interferon.Adjuvants contemplated herein include resorcinols, non-ionic surfactantssuch as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether.Enzyme inhibitors include pancreatic trypsin. Liposomes includewater-in-oil-in-water CGF emulsions as well as conventional liposomes.

Prevention of Microorganism Growth

Under ordinary conditions of storage and use, these preparations maycontain a preservative to prevent the growth of microorganisms.

The prevention of the action of microorganisms can be brought about byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thirmerosal, and the like. In manycases, it is possible to include isotonic agents, for example, sugars orsodium chloride. Prolonged absorption of the injectable compositions canbe brought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilisation. Generally, dispersions are prepared byincorporating the sterilised active ingredient into a sterile vehiclewhich contains the basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the methods ofpreparation may include vacuum drying and the freeze-drying techniquewhich yield a powder of the active ingredient plus any additionaldesired ingredient from previously sterile-filtered solution thereof.

Pharmaceutically Acceptable Carrier

As used herein “pharmaceutically acceptable carrier and/or diluent”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike. The use of such media and agents for pharmaceutical activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active ingredient, use thereofin the therapeutic compositions is contemplated. Supplementary activeingredients can also be incorporated into the compositions.

Dosage Unit Forms

It is especially advantageous to formulate pharmaceutical compositionsin dosage unit form for ease of administration and uniformity of dosage.

Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subjects to be treated; each unitcontaining a predetermined quantity of active material calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the novel dosage unitforms are dictated by and directly dependent on (a) the uniquecharacteristics of the active material and the particular therapeuticeffect to be achieved, and (b) the limitations inherent in the art ofcompounding such as active material for the treatment of disease inliving subjects having a diseased condition in which bodily health isimpaired.

The principal active ingredients are compounded for convenient andeffective administration in effective amounts with a suitablepharmaceutically acceptable carrier in dosage unit form. In the case ofcompositions containing supplementary active ingredients, the dosagesare determined by reference to the usual dose and manner ofadministration of the said ingredients.

EXAMPLES Example 1 Cell Lines and Plasmids

The cell lines MCF10A, MCF7, MDA-MB-231, Hs578T, ZR-75-1 are purchasedfrom American Type Culture Collection and maintained in the recommendedmedia except for MCF10A cells, which are cultured in DMEM supplementedwith 5% horse serum, 20 ng/ml of EGF, 0.5 μg/ml of hydrocortisone, 100ng/ml of cholera toxin, 10 μg/ml of insulin and pen/strep. BT-549 cellsare cultured in RPMI supplemented with 10% FBS. MDA-MB-435S, T-47D cellsare from Lo Ting Ling (Institute of Molecular and Cell Biology) andmaintained in DMEM supplemented with 10% FBS and 10 μg/ml of insulin.BT-20, MDA-MB-453 and BT-474 are provided by Yoshiaki Ito (Institute ofMolecular and Cell Biology). BT-20 cells are maintained in MEMsupplemented with 10% FBS. MDA-MB-453 cells are maintained in DMEM with10% FBS supplemented with 10 μg/ml of insulin. The amphotropic Phoenixpackaging cells are kindly provided by G. Nolan (Stanford University)and maintained in DMEM supplemented with 10% FBS.

The cDNAs of human TAZ is from MCG clone 19891. The full-length TAZ orFlag-tagged TAZ are constructed by polymerase chain reaction (PCR) usingthe MCG19891 clone and cloned into BamH1-Sal1 sites of the retroviralvector pBABEpuro. The Flag-tagged mouse TAZ is constructed by PCR usingmouse cDNA library from Naiyang Fu (Institute of Molecular and CellBiology) and cloned into SnaB1-Sal1 sites of the retroviral vectorpBABEhygromycin from Sofie Van Huffel (Institute of Molecular and CellBiology). pGEX-TAZ (amino acids 160-229) and pET-TAZ (amino acids160-229) are constructed by cloning the PCR-amplified cDNA fragmentencoding the indicated amino acids into EcoR1/Xho1 sites of pGEX4T-1(Amersham Biosciences) and pET32a (Novagen), respectively. pGEX-YAP(amino acids 206-262) is constructed by cloning the PCR-amplifiedfragment from human cDNA library into the EcoR1/Sal1 sites of pGEX4T-1.

Example 2 Purification of GST-Tagged Protein

One liter of E. coli BL21 (DE3) carrying the pGEX-TAZ or pGEX-YAPconstructs are grown in LB medium until OD 0.8-1 and are induced with0.1 mM of IPTG (isopropyl-β-D-thiogalactopyranoside) for overnight atroom temperature. Bacteria are harvested by centrifugation and lysed bysonication in phosphate-buffered saline (PBS). The lysate are spun at10,000 g for 30 min at 4° C. and the supernatant is mixed with 0.5 ml ofglutathione-sepharose 4B (Amersham Biosciences) for 2 h at 4° C. Thebeads are washed four times with PBS. The bound proteins are eluted with5 volumes of 10 mM reduced glutathione in 50 mM Tris (pH 8).

Example 3 Purification of His-Tagged Proteins

One liter of E. coli BL21 (DE3) carrying the pET-TAZ fusion constructsis induced as pGEX-TAZ. Bacteria are lysed by sonication in crackingbuffer (100 mM Hepes-KOH, pH7.4, 5 mM MgCl2, 500 mM KCl, 0.1%Triton-X-100, 2 mM β-mercaptoethanol and protease inhibitors cocktailfrom Roche Molecular Biochemicals). The lysate is spun and thesupernatant are collected and incubated with 1 ml of Talon MetalAffinity Resins (Clontech) for 2 h at 4° C. The resins are washed fourtimes with washing buffer (20 mM Hepes, pH7.4, 200 mM KCl, 10% glycerol,10 mM imidazole and 2 mM β-mercaptoethanol) and eluted with 5 volume ofelution buffer (20 mM Hepes, pH7.4, 200 mM KCl, 10% glycerol, 250 mMimidazole and 2 mM β-mercaptoethanol).

Example 4 Antibodies

Commercial TAZ antibody is purchased from Abcam and Imgenex. YAPantibody is purchased from Cell Signaling. Actin antibody is from Sigma.Rabbit polyclonal TAZ-specific antibodies are raised by injecting rabbitwith GST-TAZ (amino acids 160-229) three times followed by twoadditional boost injections with His-TAZ (amino acids 160-229) beforeserum is collected. The antibody is affinity purified using immobilizedHis-TAZ. To detect TAZ specifically, antibody is used in the presence of100 fold excess of GST-YAP.

Example 5 Western Blot Analysis

The indicated cells are washed once with ice-cold PBS and subsequentlylysed in ice-cold lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.3,0.25 mM EDTA, pH 8.0, 1% sodium deoxycholate, 1% Triton X-100, 0.2%sodium fluoride and 0.1% sodium orthovanadate supplemented with proteaseinhibitor cocktail). After clearance by a spin (10,000 g, 15 min), thelysate is resolved on SDS-polyacrylamide gels and blotted ontonitrocellulose membrane. The filters are blocked in 5% skimmed milk inPBS and probed with indicated primary antibody followed by horseradishperoxidase-conjugated secondary antibody (pierce). Signals arevisualized using Supersignal (Pierce).

Example 6 Retrovirus Generation and Infection

The amphotropic Phoenix packaging cells are transfected with theindicated retroviral vectors using Lipofectamine according tomanufacturer's instruction (Invitrogen). After 48 h, the retroviralsupernatants are collected, filtered (0.45 μm; Millipore) and added ontothe target cells in the presence of 5 μg/ml of polybrene (Sigma-Aldrich)for 6-8 h. Infection is done twice. After infection, the cells areselected with puromycin (1 μg/ml) for a week before being analyzed forTAZ expression by Western blotting. For re-expression of mouse TAZ inMCF-KD-715 and MCF7-KD-652 cells, the retroviral supernatants derivedfrom transfecting the amphotropic Phoenix cells withpBABEhygromycin-mTAZ are added to MCF-KD-715 and MCF7-KD-652 cells,which are selected with hygromycin (500 μg/ml) and puromycin (1 μg/ml)for a week before experiments are done.

Example 7 shRNA-Mediated Knockdown of TAZ

Short hairpin RNAs (shRNA) against human TAZ are designed using siRNAdesign program from Dharmacon and subcloned into the BgIII-Xho1 sites ofthe pSuper.Retro.puro vector (Oligoengine). Efficacy of the constructsis tested through transduction into MCF7 and Hs578T cells and Westernblot analysis of total cell lysates with the TAZ antibody. The sequencesfor the sense oligonucleotides for the knockdown construct are: KD-1,5′-GATGAATCCGGCCTCGGCGCC-3′ (SEQ ID NO: 1); KD-650,5′-AGAGGTACTTCCTCAATCA-3′ (SEQ ID NO: 2); KD-652,5′-AGGTACTTCCTCAATCACA-3′ (SEQ ID NO: 3); KD-715,5′-CAGCCTCTGAATCATATGA-3′ (SEQ ID NO: 8); KD-1331,5′-AACAAACGTTGACTTAGGA-3′ (SEQ ID NO: 9).

Example 8 Wound-Healing Assay

Cell migration is assessed in wound healing assays. Briefly, confluentMCF10A-GFP, MCF10A-TAZ, Hs578T-KD-715 and Hs578T-KD-652 cells plated ontissue culture dishes are wounded by manual scratching with 200-μlpipette tip, washed with PBS and incubated at 37° C. in complete media.At the indicated time points, phase contrast images at specific woundsites are captured.

Example 9 Anchorage-Independent Growth in Soft Agar

1.5 ml of 0.5% agar (electrograde ultra pure; Invitrogen, Carlsbad,Calif.) supplemented with RPMI, 10% FBS, are plated in six-well platesas bottom agar. Five thousand cells are mixed with 1.5 ml of 0.35% agarsupplemented with RPMI, 10% FBS, and plated on the solidified bottomagar. 1 ml of media is added on top of the solidified agar layers andthe colonies are allowed to grow in incubator at 37° C., 5% CO₂ for 2 to3 weeks. The images of cell colonies are captured with an invertedmicroscope.

Example 10 Cell Motility and Invasion Assays (Transwell Assays)

Cell motility is determined by using the 24-well chambers with 8-μm porepolycarbonate membranes (BD Biosciences). The chambers are rehydrated inserum-free medium as described by the manufacturer. Complete medium with10% FBS (750 μL) is used as chemoattractant. Suspensions of 5×10⁴ cellsin 500 μL of complete medium with 0.5% FBS are added to the inserts andincubated for 48 hours at 37° C., 5% CO₂. Cells remaining on the uppermembrane surface of the inserts are removed with a cotton swab whereasthe cells on the lower surface, as well as the ones in the wells aretrypsinized and cell number are counted.

Cell invasiveness is done essentially same as cell motility assay exceptthat the chambers used are 24-well Matrigel invasion chambers with 8-μmpore polycarbonate membranes precoated with a thin layer of MatrigelBasement Membrane Matrix (BD Biosciences).

Example 11 Tumorigenesis in Nude Mice

4- to 6-week-old female nude mice are inoculated s.c. in the left andright hind flanks or into the thoracic mammary fat pad with 5×10⁶MCF7-KD-715 or MCF7-KD-652 cells suspended in 100 μL of PBS andsimultaneously received a 60-day release pellet containing 0.72 mg ofβ-estradiol (Innovative Research of America, Toledo, Ohio). Tumordevelopment is monitored and pictures of mice are taken when the tumorsizes are bigger than 5 mm.

Example 12 Immunohistochemistry

Human breast tissue arrays (InnoGenex) are used to examine theexpression of TAZ and cytokeratin (Cam 5.2, Becton Dickinson) in normaland cancer tissues. Immunohistochemistry is performed using DakoEnvision™ System K 1395 (Dako, Carpinteria, Calif.). The slides arede-waxed in fresh xylene for 5 min for three times and re-hydratedsequentially with 100%, 95%, 80% and 75% ethanol and PBS (5 min for eachstep), followed by antigen retrieval with 2100-Retriever (PickCellLaboratories BV Prestige Medical Ltd) for 12 min in sodium citratebuffer, pH6. After cooling for 4 hr at room temperature (RT), the slidesare rinsed with water and PBS with 0.1% tween-20 before quenching with0.6% H₂O₂ in dark for 20 min. After rinsing with PBS, the slides areblocked with PBS with 5% goat serum and 2% BSA for 2 hr at RT andincubated with the primary antibody for overnight at 4° C. Subsequentlythe slides are washed with PBS with 0.1% tween-20 and followed bybiotinylated secondary antibody for 2 hr. After washing, the slides areincubated with VECTASTATIN ABC reagent for 60 min. The diaminobenzidinetetrahydrochloride (DAB) peroxidase substrate is applied to slide for3-5 min in dark and reactions are terminated by washing with PBS. Theresults are analyzed under microscope.

Example 13 TAZ Expression in Breast Cancer Cell Line

The expression of TAZ in breast cancer cell lines is examined byimmunoblot analysis using total cell lysates. Testing severalcommercially available as well as in-house generated antibodies showedthat all antibodies reacted with both TAZ and the homologous YAP. Due toefficient detection of both TAZ and YAP by a commercial antibody(IMGENEX), we have used this antibody throughout the entire study exceptfor the experiment described in FIG. 5.

The results derived from a representative experiment, shown in FIG. 1A,indicate that TAZ is expressed at varying levels in all breast cancercell lines examined. The expression levels of both TAZ and YAP arenormalized against those of actin and the quantitative results derivedfrom three independent experiments are presented in arbitrary unitsshown in FIG. 1B.

Among the breast cancer cell lines, high levels (around 4 arbitraryunits) of TAZ are detected in Hs578T, BT-549, and MDA-MB-435S cells,while moderate levels (around 2 arbitrary units) are observed inMDA-MB-231, BT-20, and T-47D cells. MCF10A, MCF7, MDA-MB-453, ZR-75-1,and BT-474 cells express low levels (around 1 arbitrary unit) of TAZ.

Significantly, three (Hs578T, BT-549, and MDA-MB-435S) of the fourhighly invasive cancer cell lines exhibit high levels of TAZ expressionwith MDA-MB-231 cells expressing moderate levels. These four cell linesare shown in Neve et al (2006) to correspond to basal-like or basal Bcancer types, representing invasive breast cancer cell types.

Most (five out of 7) of the weakly invasive cells express low levels ofTAZ with two cell lines (BT-20 and T-47D) expressing moderate levels.These results suggest that the majority of highly invasive breast cellsexpress high levels of TAZ, while the majority of weakly invasive cellsexpress low levels of TAZ.

No such correlation of YAP expression levels with invasiveness of breastcancer cells is noticed (FIG. 1B). The correlation of TAZ expressionlevel with the invasiveness of breast cancer cells suggests that TAZ maybe part of the mechanism governing the invasiveness of breast cancercells.

Example 14 Overexpression of TAZ in MCF10A Cells Induces Fibroblast-LikeMorphology and Promotes Cell Migration and Invasion

To examine the functional consequence of TAZ expression in breast cancercells, we overexpressed TAZ or Flag-tagged TAZ in MCF10A cells (whichexhibit low endogenous levels of TAZ) to levels that are about 2-3 foldof those found in high-expressing invasive cells such as Hs578T andBT-549 (FIG. 2A). This is achieved by retrovirus-mediated transduction,as MCF10A cells are not satisfactorily transfected to express exogenousproteins. Pools of MCF10A cells infected with the appropriate retrovirusare analysed to avoid clonal variations. EGFP expressing cells (leftpanels of FIG. 2B) retained the epithelial appearance seen of theparental MCF10A cells. However, cells overexpressing TAZ (right panelsof FIG. 2B) or Flag-TAZ (data not shown) developed a more reflectory andspindle-shaped fibroblast-like morphology characteristic of celltransformation.

Since TAZ expression levels correlate with the invasiveness of breastcancer cells, we examined whether TAZ overexpression could promote cellmigration and invasion. Using the wound-healing assay, we compared thecell mobility of MCF10A cells expressing TAZ relative to the cellsexpressing EGFP (FIG. 2B). The mobility of TAZ-expressing cells isdramatically enhanced. Within 14 hr, the area of wound is significantlyrecovered by the migrated TAZ-overexpressing cells. The area of wound iscompletely recovered by the migrated cells within 24 hr (right panels).In marked contrast, the wound closure of MCF10A cells expressing EGFP isnot significant within 14 hr and is only partial within 24 hr (leftpanels). The motility and invasiveness of these cells are independentlyassessed using the Transwell assay (FIG. 2C). The migration andinvasiveness of TAZ expressing cells increased about 5 and 2-3 folds,respectively, compared to control cells expressing EGFP. These resultssuggest that TAZ promotes cell migration and invasion and theseproperties may contribute to the altered morphology of cellsoverexpressing TAZ.

Example 15 shRNA-Mediated Knockdown of TAZ in MCF7 and Hs578T CellsSuppresses Cell Migration and Invasion

To verify a role for TAZ in cell migration and invasion, a complimentarybut independent approach is used that is based on RNA-interference(RNAi)-mediated knockdown of gene expression. For sustained knockdownwhile avoiding clonal variations, we analysed pools of cells stablyinfected with a retrovirus-based vector expressing shRNA targetingvarious regions of TAZ mRNA. One reported RNAi target site (KD-1) plus 4other sites of TAZ mRNA are tested for their susceptibility to knockingdown the expression of TAZ protein in MCF7 cells as assessed byimmunoblot analysis. As shown in FIG. 3A (left panel), the shRNA basedon the reported target noticeably reduced the level of TAZ protein.Among the shRNAs based on the four new targets, two (715 and 1331) hadno significant effect on TAZ protein levels, one (650) has RNAi effectcomparable to the reported one, whereas another (652) had the mostefficient effect of suppressing TAZ expression. The four shRNAsexpressing retroviruses are also used to infect Hs578T cells (rightpanel) and pools of stably transduced cells are analyzed for TAZexpression. Again, shRNA 715 and 1331 had no significant effect, whileboth shRNA 650 and 652 reduced the protein level of TAZ significantly.We therefore used cells knocked down with shRNA 652 for subsequentanalysis in comparison with cells transduced with the shRNA715, as cellstransduced with shRNA715 behaved like parental and vector-transducedcells in all analysis. Concomitant with reduced TAZ expression, theclusters of cells became more densely-packed and compact sheets of cellswith the cell density of the clusters being enhanced in the MCF7-KD-652cells as compared to MCF7-KD-715 cells (upper panels, FIG. 3B). Scanningelectron microscopy revealed that the space between cells is reduced inMCF7-KD-652 cells as compared to MCF7-KD-715 cells (lower panels, FIG.3B), resulting in the appearance of more tightly aligned/packed andcompact epithelia. This observation is obvious when cells are plated ateither low or high density cultured under standard conditions. As MCF7cells do not migrate and invade significantly using most assays, we haveanalyzed the migration and invasion using Hs578T cells. Relative toHs578T-KD-715 cells, with no detectable RNAi effect, which migratedrobustly in the wound-healing assay (left panel, FIG. 3C), knockdown ofTAZ significantly reduced the migration of Hs578T-KD-652 cells (rightpanels, FIG. 3C). The reduction of migratory and invasive abilities isalso revealed using the Transwell assay (FIG. 3D). These RNAiexperiments further bolster the conclusion derived from theoverexpression experiments that TAZ is an important regulator of cellmigration and invasion. Its expression levels correlate negatively withthe epithelial appearance of breast cells and positively with migratoryand invasive properties.

Example 16 TAZ is Important for Anchorage-Independent Growth andTumorigenesis

To examine the importance of TAZ in tumorigenesis of breast cancercells, we firstly assessed the anchorage-independent growth capabilityof TAZ-knocked-down MCF7-KD-652 cells in comparison with MCF7-KD-715cells. MCF7-KD-715 cells grew well in soft agar (upper panels, FIG. 4A),whereas the number of colonies grown in soft agar is dramaticallyreduced for MCF7-KD-652 cells. Compared to the well-developed spheres ofcell colonies of MCF7-KD-715 cells (left panel, FIG. 4B), MCF7-KD-652cells failed to grow up and only small aggregations of cell debris areobserved (right panel, FIG. 4B). These results suggest that TAZ isessential for anchorage-independent growth of MCF7. We have subsequentlyexamined whether TAZ contributes to tumorigenesis in nude mice.MCF7-KD-715 and MCF7-KD-652 cells are separately injected into the thighand fat pad of nude mice and the growth of the tumors is monitored (FIG.4C). Compared to MCF7-KD-715 cells (right side), MCF7-KD-652 cells (leftside) are compromised in forming tumors at both the thigh (left panel)and fat pad (right panel) injection sites. The quantitative analysis ofthe resulting tumors is shown in supplementary FIG. S1. These resultssuggest that TAZ is also important for in vivo tumorigenesis of MCF7cells.

Example 17 Overexpression of TAZ in Breast Cancers

To assess whether our findings obtained from analyzing the breast cancercells have physiological and clinical relevance, we have examined TAZexpression in tissue sections derived from primary breast cancers. Toovercome the cross-reactivity of anti-TAZ antibodies with YAP, wegenerated rabbit antibodies to a region of TAZ that is most divergentfrom YAP. The affinity-purified antibodies preferentially recognized TAZbut also reacted with YAP at lower efficiency (left panel, FIG. 5A).However, the antibodies recognized specifically TAZ in the presence of100 folds excess (over the antibody) of recombinant YAP fragmentcorresponding to the TAZ region used as the antigen (right panel, FIG.5A). Using this approach to specifically detect TAZ,immunohistochemistry is used to examine the primary breast cancersamples. Among 126 breast cancer samples analyzed, 27 (21.4%)overexpressed TAZ and most (21 samples) of the TAZ-expressing cancersare of invasive (infiltrating) ductal carcinomas (IDC), suggesting thatTAZ is overexpressed in a significant fraction of the IDC. Arepresentative positive labeling of TAZ in IDC is shown (right panels,FIG. 5B). The summary of our immunohistological analysis of the cancersamples is shown in Table E1 below. These results suggest that TAZoverexpression is likely to be important for the progression of breastcancer into IDC and establish the physiological and clinical relevanceof our findings with breast cancer cell lines.

TABLE E1 Analysis of TAZ Overexpression in Different Types of BreastCancers. Nor. fibroade- Medullary Breast noma LCIS IDC ILC DCIS LN metCa 0/4 0/8 2/2 21/87 3/6 0/7 1/11 0/5 Immunohistochemistry was used toexamine the expression of TAZ in each breast cancer type. The numbersshown represent the numbers of samples overexpressing TAZ in totalnumber of sample of each breast cancer type. Nor. Breast, normal breast;LCIS, lobular carcinoma in situ; IDC, invasive ductal carcinoma; ILC,invasive lobular carcinoma; DCIS, ductal carcinoma in situ; LN met,lymph node metastasis; Medullary ca, medullary carcinoma.

Example 18 TAZ Interacts with Transcriptional Factors TEAD1, TEAD2,TEAD3 and TEAD4

In vitro binding experiments with TAZ and GST-TEADs and HA-TEADs(expressed in cells) are conducted, using methods described herein andknown in the art.

In a first experiment, cell extracts derived from Hs578t cells areincubated with immobilized GST-TEAD1 (96-412), GST-TEAD2 (121-448),GST-TEAD3 (115-436), GST-TEAD4 (119-435) or GST. Proteins retained bythe beads, together with the starting material, are resolved by SDS-PAGEfollowed by immunoblotting with antibodies that react with both TAZ andYAP.

The results are shown in FIG. 8A. This figure shows that both TAZ andYAP are efficiently retained by the immobilized GST-TEADs but not GST.

In a second experiment, s578T cells stably transduced with retroviralvector to express HA-TEAD1-4 are lysed and the resulting cell lysateswere immunoprecipitated with anti-HA antibodies. The immunoprecipitates,along with starting materials, are resolved by SDS-PAGE followed byimmunoblotting using anti-HA antibody or antibodies that react with bothTAZ and YAP.

The results are shown in FIG. 8B. This figure shows that endogenous TAZand YAP (at much less efficiency) are co-immunoprecipitated withstably-expressed HA-TEAD1-4.

Example 19 Identification of TAZ Residues Involved in Binding to TEAD2

Mutant S89A of TAZ is made using standard mutagenesis techniques. Thismutant is a gain of oncogenic function mutant and is described infurther detail below.

A number of mutants of TAZ S89A are made using standard mutagenesistechniques. Among these are mutations F52A and F53A (M9) of TAZ S89A.These are designated M1-M9.

Constructs expressing FLAG tagged M1-M9 mutants are then generated.

MCF10A cells are infected with HA-TEAD2 and the Flag-tagged TEAD-bindingmutants of TAZ (FLAG-tagged M1-M9). Lysates of transfected MCF10A cellsare produced. The lysates are immunoprecipitated with anti-HA and probedwith anti-Flag antibody.

The results are shown in FIG. 9. FIG. 9 shows that residues F52 and F53(M9) in the N-terminal region of TAZ are important for interaction withTEAD2. F52A and F53A mutants are unable to interact with TEAD2.

Example 20 Mutant F53A Defective in Interaction with TEAD2 is Excludedfrom Nuclear Accumulation

MCF10A cells infected with Flag-tagged TAZ-S89A and M9 (i.e., F53A) arestained with anti-Flag antibody, using methods described here and knownto the skilled reader.

FIG. 10 shows the results. The TEAD-binding mutant fails to accumulatein nucleus. This shows that TEAD is essential for TAZ to accumulate inthe nucleus.

Example 21 Mutant F53A Defective in Interaction with TEAD2 Does NotDrive Anchorage-Independent Growth

20,000 MCF10A cells infected with either TAZ-S89A or M9 (i.e., F53A)mutant are grown in soft agar for one month and colonies are stained,using methods described here and known to the skilled reader.

FIG. 11 shows the results. The TEAD-binding mutant is unable to grow insoft-agar. This shows that interaction with TEAD is essential for TAZ toinduce oncogenic transformation.

Example 22 TAZ Induces Expression of Secreted Proteins and SurfaceMembrane Proteins

TAZ (S89A) is a mutant of TAZ generated by standard mutagenesistechniques. Serine at position 89 of TAZ is mutated to alanine. It isshown to be a gain of oncogenic function mutant.

RT-PCR is conducted on cells expressing TAZ and TAZ (S89A) to establishexpression of a number of secreted proteins and surface membraneproteins.

As shown in FIG. 12, TAZ up-regulates the expression of 8 secretedproteins (IGFBP3, ADAMTS1, CTGF, Cyr61, FSTL1, FN1, FBN1 and FBN2) aswell as 4 surface membrane proteins (AXL, ITGB2, CRIM1, and Alcam).

These proteins (including combinations of these proteins) will offer newdiagnostic biomarkers for breast and other cancers. Furthermore, thesurface membrane proteins may offer candidates for antibody therapy.

Example 23 Discussion

In our ongoing proteomics analysis of proteins in human cancer celllines, we have noticed the higher expression levels of TAZ in moreinvasive breast cancer cells. This observation prompted us toinvestigate the physiological/clinical relevance and the role of TAZ intumorigenesis of breast cancer. TAZ is widely expressed in breast cancercells. An important observation is that most highly invasive breastcancer cell lines express TAZ at levels that are about 4 times of thoseexpressed by the majority of weakly invasive breast cancer cells,implying a role for TAZ in the invasiveness of breast cancer cells. Theclinical relevance of this observation is supported by the finding thatTAZ is overexpressed in a significant fraction of breast cancers (about21.4% of 126 commercially available breast cancer samples examined). Asinvasiveness of cancer cells is dependent on increased migratory andinvasive properties, we have tested the hypothesis that the mechanism ofaction of TAZ overexpression in breast cancers and cell lines is topromote the migration and invasiveness of breast cancer cells. Both gainof function (by overexpression) and loss of function (by shRNA-mediatedknockdown) approaches are used to establish the critical role of TAZ inthe migration and invasion of breast cancer cells. Overexpression of TAZin MCF10A cells to a level about 2-3 fold of those detected in highlyinvasive cells caused a morphological change from an epithelial to afibroblast-like appearance and dramatically increased the migratory andinvasive properties of the cells. Furthermore, shRNA-mediated knockdownof TAZ expression in MCF7 and Hs578T cells reduced cell migration andinvasion. The epithelial clusters of MCF7 cells became moredensely-packed with cells when TAZ expression is knocked down. Theseresults indicate that TAZ is a negative regulator of epithelialmorphology/architecture as well as a positive regulator for invasive andmigratory behavior. It is conceivable that TAZ overexpression in breastcancer may trigger the loss of epithelial property to promote themigratory property, an important event for ductal carcinoma in situ toprogress into IDC. Finally, when TAZ expression is knocked down in MCF7cells, their anchorage-independent growth in soft agar and tumorigenesisin nude mice are retarded, suggesting that TAZ overexpression is animportant part of the process involved in breast cancer development andprogression. We believe that our experiments have addressed directly therole of TAZ rather than off-target of shRNA. Firstly, we have employedseveral different shRNAs and observed a correlation between the extentof knockdown and the observed consequence on cellular behaviors.Secondly, results derived from overexpression in MCF10A cells lead tosimilar conclusions. Finally, re-introduction of RNAi-resistant mousecDNA encoding Flag-tagged mouse TAZ (mTAZ) in MCF7-KD-652 (TAZknocked-down) significantly restored the ability of the cells to formcolonies in soft-agar and the results are shown as supplementary FIG.S2.

Although the molecular mechanism governing the function of TAZ is notfully clear, one of the mechanisms for TAZ action is to trigger a lossof epithelial morphology, to promote cell migration and invasion, and tosupport anchorage-independent growth, all of which are important forcancer initiation, progression and invasion. Although TAZ overexpressionis not sufficient to enable MCF10A cells to grow in soft agar(unpublished observation), it is important for anchorage-independentgrowth of MCF7 cells. TAZ may thus play an critical role but not besolely sufficient for anchorage-independent growth of breast cancercells. The morphological change of MCF10A cells due to TAZoverexpression is similar to the epithelial-mesenchymal transition (EMT)(21) characterized by loss of cell adhesion and increased cell mobility,whereas the altered morphology of MCF7 cells due to TAZ knockdown mightbe related to the mesenchymal-epithelial transition (MET). TAZ mightalso be part of the regulatory machinery governing the EMT/MET events inbreast epithelial cells and its de-regulated expression will enhance EMTto facilitate the development of breast cancer and invasive property.Preliminary study suggests that the expression level of E-cadherin inMCF10A and MCF7 cells is not significantly altered by eitheroverexpression or knockdown of TAZ, indicating that TAZ may regulateEMT/MET via mechanism different from those utilized by Twist, snail, andslug, which are known to promote EMT by down-regulating E-cadherin (22).Based on our current knowledge of TAZ as a co-activator of genetranscription, one possible mechanism for the action of TAZ is tointeract with other transcriptional activators to enhance thetranscription of genes that are involved in cell migration. Ourpreliminary microarray analysis seems to support this possibility asmany genes that are potentially involved in cell migration and othercellular processes are up-regulated by TAZ. We are in the process ofverifying these results to identify the genuine downstream targets ofTAZ that are involved in cell migration. At the same time, TAZ maydirectly interact with proteins that are involved in cell migration viaits PDZ domain, WW domain or coiled-coiled domain. Our preliminaryanalysis of cells expressing EGFP-tagged TAZ indicates that someEGFP-TAZ can be detected in the membrane ruffles. Further studies willbe necessary to explore these possibilities.

YAP, a protein highly homologous to TAZ, is recently identified as acandidate oncogene on the chromosome 11q22 amplicon. Overexpression ofhuman YAP in nontransformed mammary epithelial cells induces EMT,suppresses apoptosis, and promotes growth factor-independentproliferation and anchorage-independent growth in soft-agar (23). Hence,YAP and TAZ may share similar or overlapping functions. However, we didnot observe obvious correlation of YAP expression levels with theinvasiveness of breast cancer cells, whereas the expression of TAZ ismuch increased in more invasive breast cancer cell lines (FIG. 1). Inconjunction with the observation that the expression of YAP is notaffected by shRNA-mediated knockdown of TAZ, it seems that TAZ and YAPare independently-regulated. A few other studies suggest that YAP mayhave tumor-suppressing property by interacting with and stabilizingtumor suppressor p73 in the nucleus for proper execution of the celldeath pathway (24). More future studies are needed to gain fullunderstanding about these issues.

The finding that TAZ is overexpressed in breast cancers and cancer celllines and its critical role in cell migration, invasion andtumorigenesis is of significance. Firstly, it might serve as a novelbiomarker for breast cancers (especially IDCs) and our findings suggestthat a comprehensive examination of TAZ expression in a large number ofbreast cancers in terms of the prevalence, clinical outcome, andresponse to various treatments is warranted. Secondly, our findings havelaid down a novel and solid foundation for future studies aiming toreveal additional insights into the molecular mechanism governing itsrole in breast cancer cell migration, invasion and tumorigenesis and itsinterplay with other proteins involved in the development, progressionand metastasis of breast cancers. Furthermore, TAZ might offer a noveltarget to treat breast cancers as its expression is preferentiallyincreased in invasive breast cancer cells and the levels correlate withinvasiveness. Because TAZ plays an important role in tumorigenesis ofbreast cancer cells, yet clearly not essential for mouse development andfertility, it might be an effective yet selective target for breastcancer therapy.

REFERENCES

-   1. Hinestrosa M C, Dickersin K, Klein P et al. Shaping the future of    biomarker research in breast cancer to ensure clinical relevance.    Nat Rev Cancer 2007; 7:309-315.-   2. Sjoblom T, Jones S, Wood L D et al. The consensus coding    sequences of human breast and colorectal cancers. Science 2006;    314:268-274.-   3. Allred D C, Brown P, Medina D. The origins of estrogen receptor    alpha-positive and estrogen receptor alpha-negative human breast    cancer. Breast Cancer Res 2004; 6:240-245.-   4. Zajchowski D A, Bartholdi M F, Gong Y et al. Identification of    gene expression profiles that predict the aggressive behavior of    breast cancer cells. Cancer Res 2001; 61:5168-5178.-   5. Thompson E W, Paik S, Brunner N et al. Association of increased    basement membrane invasiveness with absence of estrogen receptor and    expression of vimentin in human breast cancer cell lines. J Cell    Physiol 1992; 150:534-544.-   6. Sommers C L, Byers S W, Thompson E W, Torri J A, Gelmann E P.    Differentiation state and invasiveness of human breast cancer cell    lines. Breast Cancer Res Treat 1994; 31:325-335.-   7. Price J E, Polyzos A, Zhang R D, Daniels L M. Tumorigenicity and    metastasis of human breast carcinoma cell lines in nude mice. Cancer    Res 1990; 50:717-721.-   8. Neve R M, Chin K, Fridlyand J et al. A collection of breast    cancer cell lines for the study of functionally distinct cancer    subtypes. Cancer Cell 2006; 10:515-527.-   9. Debnath J, Muthuswamy S K, Brugge J S. Morphogenesis and    oncogenesis of MCF-10A mammary epithelial acini grown in    three-dimensional basement membrane cultures. Methods 2003;    30:256-268.-   10. Kanai F, Marignani P A, Sarbassova D et al. TAZ: a novel    transcriptional co-activator regulated by interactions with 14-3-3    and PDZ domain proteins. EMBO J 2000; 19:6778-6791.-   11. Park K S, Whitsett J A, Di Palma T, Hong J H, Yaffe M B,    Zannini M. TAZ interacts with TTF-1 and regulates expression of    surfactant protein-C. J Biol Chem 2004; 279:17384-17390.-   12. Cui C B, Cooper L F, Yang X, Karsenty G, Aukhil I.    Transcriptional coactivation of bone-specific transcription factor    Cbfa1 by TAZ. Mol Cell Biol 2003; 23:1004-1013.-   13. Tian Y, Li D, Dahl J, You J, Benjamin T. Identification of TAZ    as a binding partner of the polyomavirus T antigens. J Virol 2004;    78:12657-12664.-   14. Mahoney W M, Jr., Hong J H, Yaffe M B, Farrance I K. The    transcriptional co-activator TAZ interacts differentially with    transcriptional enhancer factor-1 (TEF-1) family members. Biochem J    2005; 388:217-225.-   15. Murakami M, Nakagawa M, Olson E N, Nakagawa O. A WW domain    protein TAZ is a critical coactivator for TBX5, a transcription    factor implicated in Holt-Oram syndrome. Proc Natl Acad Sci USA    2005; 102:18034-18039.-   16. Murakami M, Tominaga J, Makita R et al. Transcriptional activity    of Pax3 is co-activated by TAZ. Biochem Biophys Res Commun 2006;    339:533-539.-   17. Hong J H, Hwang E S, McManus M T et al. TAZ, a transcriptional    modulator of mesenchymal stem cell differentiation. Science 2005;    309:1074-1078.-   18. Hong J H, Yaffe M B. TAZ: a beta-catenin-like molecule that    regulates mesenchymal stem cell differentiation. Cell Cycle 2006;    5:176-179.-   19. Hossain Z, Ali S M, Ko H L et al. Glomerulocystic kidney disease    in mice with a targeted inactivation of Wwtr1. Proc Natl Acad Sci    USA 2007; 104:1631-1636.-   20. Tian Y, Kolb R, Hong J H et al. TAZ promotes PC2 degradation    through a SCFbeta-Trcp E3 ligase complex. Mol Cell Biol 2007;    27:6383-6395.-   21. Maeda M, Johnson K R, Wheelock M J. Cadherin switching:    essential for behavioral but not morphological changes during an    epithelium-to-mesenchyme transition. J Cell Sci 2005; 118:873-887.-   22. Peinado H, Olmeda D, Cano A. Snail, Zeb and bHLH factors in    tumour progression: an alliance against the epithelial phenotype?    Nat Rev Cancer 2007; 7:415-428.-   23. Overholtzer M, Zhang J, Smolen G A et al. Transforming    properties of YAP, a candidate oncogene on the chromosome 11q22    amplicon. Proc Natl Acad Sci USA 2006; 103:12405-12410.-   24. Strano S, Blandino G. YAP1 meets tumor suppression. Mol Cell    2007; 27:863-864.-   Hong, J.-H.; Hwang, E. S.; McManus, M. T.; Amsterdam, A.; Tian, Y.;    Kalmukova, R.; Mueller, E.; Benjamin, T.; Spiegelman, B. M.;    Sharp, P. A.; Hopkins, N.; Yaffe, M. B.: TAZ, a transcriptional    modulator of mesenchymal stem cell differentiation. Science 309:    1074-1078, 2005. PubMed ID: 16099986-   Kanai, F.; Marignani, P. A.; Sarbassova, D.; Yagi, R.; Hall, R. A.;    Donowitz, M.; Hisaminato, A.; Fujiwara, T.; Ito, Y.; Cantley, L. C.;    Yaffe, M. B.: TAZ: a novel transcriptional co-activator regulated by    interactions with 14-3-3 and PDZ domain proteins. EMBO J. 19:    6778-6791, 2000. PubMed ID: 11118213-   Murakami, M.; Nakagawa, M.; Olson, E. N.; Nakagawa, O.: A WW domain    protein TAZ is a critical coactivator for TBX5, a transcription    factor implicated in Holt-Oram syndrome. Proc. Nat. Acad. Sci. 102:    18034-18039, 2005. PubMed ID: 16332960-   Polyak K. On the birth of breast cancer. Biochim Biophys Acta. 2001    1552(1):1-13. Review-   Singapore Cancer Registry Report No. 5 “Cancer Incidence in    Singapore, 1993-1997” published in the Yr 2000-   Neve et al (2006). A collection of breast cancer cell lines for the    study of functionally distinct cancer subtypes. Cancer Cell 10,    515-527.-   Sudol M, Bork P, Einbond A, Kastury K, Druck T, Negrini M, Huebner    K, Lehman D. (1995). Characterization of the mammalian YAP    (Yes-associated protein) gene and its role in defining a novel    protein module, the WW domain. J Biol Chem. 1995 Jun. 16;    270(24):14733-41.

Each of the applications and patents mentioned in this document, andeach document cited or referenced in each of the above applications andpatents, including during the prosecution of each of the applicationsand patents (“application cited documents”) and any manufacturer'sinstructions or catalogues for any products cited or mentioned in eachof the applications and patents and in any of the application citeddocuments, are hereby incorporated herein by reference. Furthermore, alldocuments cited in this text, and all documents cited or referenced indocuments cited in this text, and any manufacturer's instructions orcatalogues for any products cited or mentioned in this text, are herebyincorporated herein by reference.

Various modifications and variations of the described methods and systemof the invention will be apparent to those skilled in the art withoutdeparting from the scope and spirit of the invention. Although theinvention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the claims.

The invention claimed is:
 1. A method of treating breast cancer in anindividual, the method comprising administering to said individual anagent that down-regulates the expression, amount or activity of TAZ,wherein said administering renders a cell of said breast cancernon-cancerous, non-invasive or non-metastatic, and wherein said agentcomprises an siRNA or shRNA that down-regulates TAZ expression via RNAinterference comprising a sequence selected from shRNA 1 (senseoligonucleotide sequence 5′-GATGAATCCGGCCTCGGCGCC-3′), shRNA 650 (senseoligonucleotide sequence 5′-AGAGGTACTTCCTCAATCA-3′) or shRNA 652 (senseoligonucleotide sequence 5′-AGGTACTTCCTCAATCACA-3′) wherein U issubstituted for T.
 2. The method of claim 1, wherein the breast canceris an invasive ductal carcinoma (IDC).